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Report into the epidemiology and control of an outbreak of infectious salmon anaemia in the Shetland Islands, Scotland

Authors:
  • Scottish Government Marine Directorate
Scottish Marine and Freshwater Science Volume 1 No 4
Report into the epidemiology and control of an outbreak
of infectious salmon anaemia in the Shetland Islands,
Scotland
A G Murray, L A Munro, I S Wallace, M Hall, D Pendrey, D
I Fraser, B Berx, E S Munro, C E T Allan, M Snow, R
McIntosh, D W Bruno, P A Noguera, D Smail & R S
Raynard
© Crown copyright 2010
This report represents the views of the authors and has not been subject to
a full peer review process
Scottish Marine and Freshwater Science Vol 1 No 4
Report into the Epidemiology and Control of an Outbreak of
Infectious Salmon Anaemia in the Shetland Islands, Scotland
A G Murray, L A Munro, I S Wallace, M Hall, D Pendrey, D I Fraser, B
Berx, E S Munro, C E T Allan, M Snow, R McIntosh, D W Bruno, P A
Noguera, D Smail and R S Raynard
Published by Marine Scotland – Science
ISSN: 2043-7722
Report into the Epidemiology and Control of an Outbreak of Infectious
Salmon Anaemia in the Shetland Islands, Scotland
Alexander G. Murray, Lorna A. Munro, I. Stuart Wallace, Malcolm Hall, Daniel
Pendrey, David I. Fraser, Barbara Berx, Eann S. Munro, Charles E.T. Allan, Mike
Snow, Rebecca McIntosh, David W. Bruno, Patricia A. Noguera, David Smail and
Rob S. Raynard
Marine Laboratory, Marine Scotland Science
375 Victoria Road, Aberdeen AB11 9DB
Summary:
An outbreak of infectious salmon anaemia (ISA) in the Scottish Shetland Islands
during 2008/9 is described during which six sites were confirmed ISAV positive.
Spread of the virus via movement of fish between marine sites, harvest vessels,
movements of smolts and wild fish appear to have been of little or no importance.
The spread is associated with hydrodynamic currents, although local intra-company
activity may have caused some spread. The application of a statutory control
strategy by Marine Scotland, based on the use of its established model (Anon 2000)
has apparently limited the occurrence and economic impact of ISA to management
area 3a; however spread within this area has been extensive. This localised water-
borne spread is in contradiction to a previous outbreak in 1998/9 which was spread
over a wide geographic area by transport of fish and harvest vessels. The
development of industry codes of practice and good biosecurity procedures, following
the 1998/9 outbreak, that limited marine site-to-site movement of live fish and
improved disinfection of vessels and processing plant waste, may explain why the
2008/9 spread of infection was localised. Depopulation of confirmed sites is a key
element of eradication and this was achieved within 7 weeks of confirmation,
although the last confirmed case suggests subclinical infection may persist
undetected for months. The potential sources of ISAV infection that were
investigated did not determine the origin of the 2008/9 outbreak. Local evolution
from an avirulent strain of ISAV; importation of ova; or association with movement of
equipment could have caused the outbreak. The virus responsible for the 2008/9
outbreak belongs to a different genogroup (group 1) to the 1998/9 virus (group 3).
The intensive cultivation of salmon farming, close proximity of sites and historic
absence of synchronous fallowing of management areas is considered to have
increased the risk of disease outbreaks and their re-emergence, such as ISA in the
Shetland Islands. A policy of synchronous fallowing and stocking of sites within
management area 3a is being considered by industry in consultation with Marine
Scotland to address this risk. Where movement of fish occurs between sites in
1
different management areas, this represents the greatest risk of regional-scale
spread of diseases such as ISA. Controls appear to have been effective in
minimising that risk.
1. Introduction
Infectious salmon anaemia (ISA) is a highly infectious orthomyxoviral disease of
farmed Atlantic salmon (Salmo salar) caused by ISA virus (ISAV) which is notifiable
to the World Organisation for Animal Health (OIE 2009). Clinical ISA has caused
major losses to salmon farmers in six countries (OIE 2009). The disease was first
reported from Norway in 1984 (Thorud and Djupvik 1988), where it is still widespread
(Lyngstad et al. 2008). Subsequently it occurred in Canada, the USA, the Faeroe
Islands and Chile (Mardones et al. 2009). None of these 5 territories have officially
eradicated ISA (Mardones et al. 2009). There are no recent reports of ISA from the
Faeroes, but vaccination is practiced and vaccinated salmon may be carriers of the
virus (OIE 2009).
An outbreak occurred in Scotland in 1998/9 (Anon 2000; Stagg et al. 2001; Stagg
2003) and this was eradicated at a cost then of over £20M (Hastings et al. 1999).
During this outbreak, ISAV was confirmed on 11 sites with an additional 34 suspect
sites being identified which were scattered across virtually the entire region of
Scottish salmon farming before its eventual eradication (Stagg 2003). At the time of
the outbreak, native Scottish wild salmonids were screened for ISAV and evidence of
infection, but not ISA disease, was found in Atlantic salmon, brown and sea trout
(both Salmo trutta) (Raynard et al. 2001).
The ISA virus can infect salmonid species such as brown trout, sea trout and rainbow
trout (Oncorhynchus mykiss), which can become carriers but do not suffer clinical
disease (Nylund and Jakobsen 1995). Both the freshwater brown and anadromous
sea trout are native to Scotland and although rainbow trout are non native they are
sometimes found in the wild due to stocking for angling purposes or as fish farm
escapes. The virus has also been reported to be replicated in non-salmonid species
such as herring (Clupea harengus) (Nylund et al. 2002) and Atlantic cod (Gadus
morhua) (Grove et al. 2007) although these reports relate to experimental challenges
rather than to wild infections.
2
Infectious salmon anaemia does not represent a risk to human health since the virus
does not grow at temperatures above 25 ºC and is inactivated at 37 ºC and therefore
unable to survive at human body temperatures (SANCO/C3/AH/R18/2000).
This report describes a second outbreak of ISA in Scotland that occurred during
2008/9. This has been geographically confined to a relatively small area in the
southwest of the Shetland Islands, (Anon 2009a). Six infected sites were confirmed
and depopulated; the management area (MA) then underwent a synchronised
fallowing. However, controls on the MA remain in place until a 2 year programme of
surveillance and testing has been completed to allow the Shetland Islands MA 3a to
be re-declared as part of the UK ISA free zone. Scottish Government policy is to
confine and eradicate ISA, in accordance with EU Council Directive 2006/88/EC, and
to take appropriate measures to regain and maintain its disease-free status.
3
2. Background
2.1. Farm Distribution of ISA Susceptible Fish
Figure 1 A map of Management Area 3a (MA 3a) inserted into a map of Scotland.
The entire MA is in the surveillance zone, while the control zones were based on
sites within a tidal excursion distance of confirmed or suspect sites. The initial
control zone was placed around site A, the origin of infection, followed by two
suspect sites, and expanded as further cases were confirmed.
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In 2008 Scotland produced 128,606 tonnes of farmed Atlantic salmon from 257
marine sites and 2628 tonnes of sea-farmed rainbow trout from 9 sites (Walker
2009). A further 311 tonnes of brown and sea trout were also produced but separate
statistics are not published for marine and freshwater farms. A third of Scottish
salmon production, 42,593 tonnes, came from the Shetland Islands (Walker 2009) in
the far north of Scotland (60 ºN) (Figure 1).
Scottish salmon production is divided for the purposes of disease control, including
ISA, into MAs (Anon 2009b), which are defined on the basis of overlapping tidal
excursion zones. These MAs use circles with radii of 3.6 km in Shetland (7.2 km
elsewhere in Scotland) based partly on a simple but robust tidal model (Stagg 2003).
A MA is defined by all sites with overlapping radii and stretches until a break occurs
that is large enough that the circles do not overlap (i.e. a separation distance of > 2 ×
3.6 km). These MAs were originally devised as part of the controls for the 1998/9
ISA outbreak (Stagg 2003) and simple modelling indicates they are likely to be
appropriate for ISA control, if not for other pathogens (Murray et al. 2005).
In 2008/9 ISA cases were restricted to one MA in south west Shetland (MA 3a)
(Figure 1). This MA contained: 42 registered finfish farm sites on which controls
were set; 16 inactive sites; 11 fallow prior to the outbreak; 13 sites which were
populated with salmonids at the time of the outbreak (having not yet been harvested),
and 2 sites producing non-susceptible species (2006/88/EC Part II, Annex IV), one
containing halibut (Hippoglossus hippoglossus) and the other, a small research site,
holding both ballan and goldsinny wrasse (Labrus bergylta and Ctenolabrus
rupestris). The MA is the largest in Scotland (Anon 2009b) and produced over 10%
of Scotland’s farmed salmon in 2007. The MA also contains a fish processing plant
situated at the port of Scalloway.
2.2. Detection of ISAV
2.2.1. Surveillance
Fish Health Inspectors (FHIs) from the Marine Scotland Marine Laboratory carry out
routine disease surveillance at fish farms in accordance with current legislation,
including EU Council Directive 2006/88/EC and The Aquatic Animal Health
(Scotland) Regulations 2009 and in accordance with the Fish Health Inspector’s
charter. However, the putative index case of the 2008/9 outbreak (i.e. the site at
which the pathogen was first found) was detected as a result of intelligence-led
surveillance when the site, amongst others, were inspected following reports from the
Shetland mainland of health problems, large scale mortality and disposal of dead
fish. These losses were attributed to sea lice (Lepeophtheirus salmonis) by industry
and subjected to further investigation by the FHI. As part of these investigations, an
inspection at site A was undertaken on 9th December 2008, when FHIs took
diagnostic samples from 5 moribund fish and on obtaining results indicating ISAV
5
infection, returned on 16th December to take a further statutory sample of 150 fish.
Following initial investigations and site inspections, at that point in time, this site was
the only site at which positive test results for ISAV were obtained.
Following confirmation of ISA on this site on 2nd January 2009, MA 3a became a
surveillance zone, and within this all sites within 3.6 km of confirmed or suspect sites
became a control zone (Figure 1). Marine Scotland FHIs visited sites within MA 3a
on a monthly basis and collected records of site mortality levels. Initially 150 fish
samples were taken at all sites within the control zone thereafter monthly inspections
were conducted, while bi-monthly inspections were undertaken at all sites within the
outlying surveillance zone. If clinical, and/or gross signs of disease were evident the
sites were subject to 5 to 10 fish full diagnostic sampling. If results indicated possible
infection and suspicion of disease on site, but test results did not confirm ISA, a
further 150 fish sample was collected. Confirmation was possible from the diagnostic
sample when results were obtained in accordance with EU directives. Sites in other
MAs that had possible past contacts with MA 3a, including sites from which fish had
been moved to or from MA 3a and a site near a processing plant (fish obtained from
MA 3a had been held in a cage at the plant prior to processing), were also subjected
to 150 fish tests and bimonthly inspections. Where direct contact was made with
control zone sites, these sites were subjected to a 150 fish test.
Sites that receive fish from ISA confirmed sites (before these were confirmed and
further movement therefore banned) are automatically classified as officially suspect.
Passive surveillance methods (where the official service follows up reports of
possible disease e.g. from farmers or vets (Hadorn et al. 2008)) were used to
enhance surveillance when one site was re-visited ahead of schedule after reports by
a veterinarian of possible ISA and elevated mortalities. As ISA is a notifiable disease
there is a legal obligation on anyone with reason to suspect its presence on a fish
farm to report this suspicion to the competent authority (FHI) (The Aquatic Animal
Health (Scotland) Regulations 2009).
2.2.2 Diagnosis of ISA
Diagnosis of ISA was carried out in accordance with EU legislation, EC/91/67
followed by EC/2006/88, as implemented by current regulation; following European
Law, Commission Decision 2003/466/EC, and following the OIE (2009). A health
inspection is a visit by an official service for the purpose of conducting health checks
on a farm or zone. This involved: clinical observation, by qualified and experienced
fish health inspectors, of gross pathology associated with fish disease; prompting a
diagnostic investigation which includes approved laboratory diagnostic tests as
provided for in regulation, involving light microscopy; quantitative Real-time
polymerase chain reaction (qPCR) and virus isolation processed by the diagnostic
6
laboratories on samples taken by FHI. Additional diagnostic methods were used,
where appropriate, as approved by the OIE and EU law (Table 1).
Diagnostic methods are detailed in Appendix 1, and summarised in the following text.
A positive qPCR result (Snow et al. 2006; 2009) in the 5-10 fish diagnostic sample
does not automatically result in ISA confirmation, or even official suspicion,
nevertheless this triggers the collection of a further 150 fish sample. If gross
pathological signs of ISA, such as dark liver, are present in combination with qPCR
positive results then the site will be placed under official suspicion, likewise qPCR
results can be confirmed by indirect tissue imprint fluorescent antibody test (IFAT)
(Stagg et al. 1999) or histology. Virus isolation, with virus identified using TO cell
susceptibility (Wergeland and Jakobsen 2001) plus IFAT (Falk et al. 1998), alone is
enough to result in the confirmation of ISA even if unsupported by other diagnostic
tests, provided there are at least two independent isolations. Confirmation was made
following a case review by senior diagnosticians, the leader of the FHI and the
programme director.
Table 1
Evidence use to provide official confirmation of ISA
Test 1 Test 2 Result
Virus isolation Confirmed
qPCR Confirmed
Mortality, clinical signs and pathology (e.g.
microscopy) consistent with ISA
IFAT Confirmed
Virus isolation from 2 independent
samples
Confirmed
qPCR Confirmed Virus isolation
IFAT Confirmed
Diagnostic tissue samples obtained from farmed fish for qPCR and virus isolation
were pooled using tissue from 5 individuals to optimise diagnostic costs. Diagnostic
samples may consist of a few moribund individuals and in such cases smaller pools
or individual fish were tested. As part of the surveillance wild fish were also sampled
(Wallace et al. 2009), freshwater fish were not pooled owing to the smaller numbers
available while marine fish were pooled but sometimes pools of <5 fish were used
because the numbers of the different species caught were not always divisible by 5;
fish of different species were not pooled together.
Using methods described by Nérette et al. (2008), virus isolation was found to have a
specificity (Sp) of 100% for ISA but is slower, taking up to 4 weeks, and has less
sensitivity (Se) than qPCR; isolation Se = 88.7% as opposed to 93% for qPCR
(Nérette et al. 2008). The qPCR was found to be only 99% specific, which means
significant numbers of false positive results are possible when large numbers of fish
are sampled. For this reason unsupported qPCRs are treated as false positives if
they could not be confirmed by other indicators of ISA. Positive qPCR results can be
7
generated by detection of the putatively avirulent HPR0 strains of ISAV (McBeath et
al. 2009) or possibly even by genetic material from totally unrelated organisms
(Gregory et al. 2007), although in this case a different Real Time-PCR test was used.
These values of Se and Sp may differ slightly in Scotland as diagnostic methods and
the viruses are slightly different. However, differences are likely to be small and a set
of samples have specifically been collected (data not discussed here) that will allow
future estimates to be made for Scotland using the same analysis as Nérette et al.
(2008).
During the laboratory analysis the qPCR diagnostic tests are confirmed using three
replicate reactions taken from one homogenised sample of material. Only if all three
replicates are positive is the test positive. This replication increases Sp with respect
to errors, but does not correct for a systematic false positive, such as presence of
HPR0. The requirement of replication could potentially reduce diagnostic sensitivity
when viral titre (concentration in the tissue) is close to the theoretical limit of
detection by qPCR.
The examination of stained tissue sections provides unique information regarding
tissue changes that will include ISA as well as any other infectious or non-infectious
co-infection. For this sections from the gut; heart; spleen; kidney; liver; pancreas and
skeletal muscle were stained with haematoxylin and eosin and examined by light
microscopy.
The ISAV responsible for the outbreak, 2008-0982, was isolated and its
haemagglutinin-esterase protein gene sequenced. This has been compared with the
sequence from other EU-G1 isolates, including the 1998-390 virus that was isolated
in the previous Scottish outbreak in 1998/9.
2.2.3 Response to Confirmation of ISA
On suspicion of ISA, a thirty day notice was placed on the suspect site A, 12
December 2008, thereby imposing movement restrictions for this period. On
confirmation of ISA at the index site the Scottish Government imposed restrictions
preventing movement into and out of MA 3a, which formed control and outlying
surveillance zones (Anon. 2009b) and Figure 1). Within the control zone (3.6 km
from a suspect or confirmed site) inspection was increased with monthly visits and
reports of mortality from sites. The area within 3.6 km of the index site, and
subsequently of other suspect or confirmed sites, formed the control zone, which
thus expanded as further sites were confirmed (Figure 1). Scottish Government
policy is for rapid removal of all fish from ISA confirmed sites in accordance with EU
Council Directive 2006/88/EC. Fish of harvestable size, not displaying external signs
of clinical ISA disease, as observed individually at the point of humane killing of the
fish, were taken for processing at an approved biosecure plant. Fish that were
undersized or displaying gross disease signs were discarded and disposed of as
8
waste material, by an approved manner, under current waste disposal legislation.
Historically in Scotland (during the previous ISA outbreak in 1998-99), confirmed
sites were fallowed for 6 months while those in the control zone fallowed
synchronously for 3 months (Stagg 2003). All sites in the surveillance zone were
fallowed for 6 weeks but synchronisation was not achieved. As the fallowing period
in 2009-10 was synchronous within the control zone, it started when the last site in
the area has been cleared of fish. Further details, including notification procedures
are provided in Appendix 3.
2.3. Investigating the Potential Routes of Spread
The hazards associated with the risk of spread of infection were investigated. Risk
assessment, results and analysis are described in section 3.3.
2.3.1. Movements of Fish and Harvest Vessels
Marine Scotland Science provides advice to Marine Scotland Policy on the
movements of fish between sites under control and also collects data on such
movements (Munro and Gregory 2009). Live fish movements are recognised to be
among the most effective ways of spreading pathogens (Murray and Peeler 2005)
and therefore these records have been analysed to determine if the movements of
fish between sites within MA 3a and between sites in this MA and other MAs
contributed to spread of ISAV.
Vessels that deliver fish to processing plants can spread infection, including ISAV,
when they return from the processing plant (Murray et al. 2002; Munro et al. 2003).
Two processing plants were used to process fish produced at sites within MA 3a, one
at Scalloway, Scalloway Processing Plant (SPP) within MA 3a, and the other in
Lerwick, Lerwick Processing Plant (LPP) on the east coast of Shetland (Figure 7).
The FHIs obtained records from the logs of vessels involved in transporting fish
between sites in MA 3a and these processing plants. They also visited the
processing plants to assess biosecurity and disinfection regimes, as it would require
a failure of biosecurity to contaminate visiting vessels causing them to become
vectors.
2.3.2. Vertical Transmission
There is currently scientific disagreement on the occurrence of vertical transmission
of ISAV (Lyngstad et al. 2008; Vike et al. 2009). Historically ISAV was not
considered to be vertically transmitted but in light of the current debate which does
not rule out the option of vertical transmission, it is important to consider its potential
effects. If vertical transmission did occur then this could be significant both as a
potential source of the Shetland outbreak, since imported ova are used, and as a
potential route of spread within Scotland and beyond via broodstock produced in
9
Shetland. It is also possible that ISAV could be horizontally transmitted e.g. on the
outside of poorly disinfected eggs (pseudovertical transmission). We therefore
assess sources of input of smolts to Shetland and conduct a risk assessment on the
potential exposure of ova produced from broodstock within Shetland.
2.3.3. Wild Fish
It is possible that ISAV may be transmitted by movements of wild fish (Nylund and
Jackobsen 1995; Raynard et al. 2001). During this outbreak two wild fish surveys
were undertaken by Marine Scotland (Wallace et al. 2009), one of wild marine fish
from within MA 3a, the second of fish from fresh waters draining into the MA (Figure
2).
The wild marine fish survey was conducted at 3 locations (Figure 2) and consisted of
multiple trawls to gain good numbers of fish for screening. These trawls were carried
out between the 9 and 11 February 2009. Samples were pooled in groups of 5 fish,
but organs were collected individually (kidney, gill, brain and heart), e.g. a pool of
heart material from 5 fish.
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Figure 2 Wild fish sampling locations for the ISA investigation: = fish farms in MA
3(a); dotted lines are numbered marine tows; solid lines are numbered electrofishing
sites and numbers refer to the catch breakdown tables.
The freshwater fish were sampled from 3 burns (1 sampled over 2 days) on the 23 –
25 March. The slightly later date of sampling was chosen to allow the freshwater
temperatures to increase making the fish more active and easier to catch (personal
experience). Additionally this would increase the chances of catching migratory sea
trout returning to freshwater at this time of year. Fish were caught using
electrofishing and the fish were sampled individually, with the same organs collected
as for wild marine fish.
In addition, 2 saithe (Pollachius virens) found in a cage on an ISA infected salmon
farm were opportunistically sampled taking the same tissues as listed above.
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2.3.4. Hydrodynamic Spread
The ISAV is transmitted via water even from fish that are not displaying clinical
disease signs (Totland et al. 1996). However, shedding increases 2 days before
death (Gregory et al. 2009) so populations with a high prevalence of clinically
infected fish represent a high risk of pathogen spread.
Several studies have shown that sites in proximity to infected sites are more likely to
become infected. Jarp and Karlsen (1997) identified an enhanced risk of infection if
infected farms (odds ratio (OR) 8, Kelsey et al. 1996) or slaughterhouses without
disinfection (OR 14.6) were within 5 km of sites, with the odds increasing as the
distance decreased. Modelling by Scheel et al. (2007) found infected sites within 5
km to be of comparable risk to sites with shared ownership. Lyngstad et al. (2008)
found that infected sites within 10 km were likely to share ISA viruses of the same
genogroup indicating possible shared infection. Aldrin et al. (2010) showed risk of
infection increased rapidly with inverse distance from the nearest infected neighbour,
provided this was <10 km away. Using currents estimated from a hydrographic
model Gustafson et al. (2007) observed that sites down current from an infected site
were more likely to pick up infection than those situated up current.
The spatial pattern of the outbreak was assessed to consider possible spread of
pathogen amongst sites, caused by water movements. It is important to consider the
role of infected sites within a tidal excursion of adjacent uninfected sites and the risks
of transmission. Within management area 3A there was a high concentration of farm
sites within close proximity to each other, enhancing the potential risk of
hydrographic and biological interaction amongst them.
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3. Results and Discussion
3.1. Chronology of the Outbreak
At the time that the putative index case was confirmed (2 January 2009) there were
12 stocked Atlantic salmon farms one trout farm and two non-susceptible species
farms in MA 3a. The site testing history is summarised (Table 2); only qPCR and
virology results from the last diagnostic test taken from each site are listed. Other
results available for some cases but not listed here included immunohistochemistry
(IHC); IFAT; histology and clinical disease signs. A complete list of diagnostic test
results from samples taken from diagnostic, statutory or research purposes is
included in Appendix 2. Due to the long time period required to obtain a virology
result, sites D, E, and F were confirmed by other positive diagnostic tests as
described in the following text. When the virology results were obtained, from earlier
diagnostic tests or statutory 150 fish tests, these positive results supported
confirmation. Tests that produced negative diagnostic results may have occurred
earlier than the results listed in Table 2. One sample (from site F) generated a
positive qPCR that was not confirmed as there were no clinical or gross signs of ISA
and no other positive diagnostic test results, until 10 months later when the site did
test positive during harvesting.
The putative index case site A was visited on 9 December 2008 as part of an
investigation into fish health problems ascribed to sea lice within MA 3a. Diagnostic
sampling resulted in a positive ISAV qPCR so the site was re-visited and 150 fish
were sampled in 30 pools. The results of testing confirmed the presence of ISAV by
qPCR (8/30) and virology (7/30) on the 2 January 2009. Harvesting was ongoing
and the site was fallowed by 20 December (Figure 3.I). However, at the
management area level, movement restrictions and the previously outlined enhanced
surveillance regime were implemented.
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Table 2
Chronology of the ISA outbreak illustrating the date of a positive sample or last
negative sample before harvest. Only qPCR and virology results (in pools of 5 fish)
are listed, other results by IHC, IFAT, and histology and diagnostic signs were also
available for some cases. Sites harvested before testing began are not listed in
table. The number of fish on site is that at the end of 2008, except site A for which
this is the number before harvesting began.
Site Owner Species Last Date
Sampled
Date
Confirmed
Test Result Date
Depopulated
Fish on
site
A C1 Salmon 16/12/2008 02/01/2009 PCR 8/30
V 7/30
20/12/2008 87,000*
B C2 Salmon 12/01/2009 30/01/2009 PCR 4/30
V8/30
06/03/2009 316,000
C C2 Salmon 18/03/2009 20/03/2009 PCR 16/30
V15/30
08/04/2009 263,000
D C2 Salmon 14/05/2009 18/05/2009 PCR 1/1
V1/1
02/07/2009 284,000
E C3 Salmon 19/05/2009 21/05/2009 PCR 25/30
V26/30
27/05/2009 6,000
F C4 Salmon 13/01/2009 30/10/2009 PCR 1/1
V 1/1
26/11/2009 286,000
g C1 Salmon 06/01/2009 N/A PCR 0/30
V 0/30
07/01/2009 297,000
h C1 Salmon 08/01/2009 N/A PCR 0/2 14/01/2009 178,000
i C2 Salmon 14/01/2009 N/A PCR 0/30 16/01/2009 301,000
j C1 Salmon 17/01/2009 N/A PCR 0/30 20/01/2009 249,000
k C4 Salmon 16/01/2009 N/A PCR 0/30 28/01/2009 60,000
l C2 Salmon 13/01/2009 N/A PCR 0/30 25/02/2009 250,000
m C5 Trout 29/01/2009 N/A PCR 0/30 02/04/2009 142,000
n C6 Halibut N/A N/A N/A 27/04/2009 6,000
o C3 Wrasse N/A N/A N/A N/A 380
The movement restrictions imposed when the index case was confirmed prevented
any movement of fish into or out of the surveillance zone, although movement of live
fish in sealed well boats for harvesting at LPP were authorised following a risk
assessment, ensuring good biosecure practices (see later).
Two sites had received fish from the putative index case (site A) and for this reason
were automatically placed under official ISA suspicion. One of these sites had
already been harvested and the other (site g) was subject to a 150 fish test which
was negative by both qPCR and virology. The last transport of live fish from A to g
had occurred on 27 June 2008 approximately six months before ISA was detected at
the index site A. This strongly suggests ISA was absent or, if present, at a very low
level on site A in June, i.e. ISA emerged within the last six months of 2008. While it
is possible that another site was the undetected true index case, the observations
14
strongly suggest ISA had emerged recently, which has important implications for the
source and potential spread of the outbreak.
Figure 3 Maps of MA 3a on: (I) 31st December 2008; (II) 31st January 2009; (III) 31st
March 2009 and (IV) 25th May 2009. The letters refer to the farms listed in Table 2:
= farms depopulated prior to the outbreak; = uninfected farms; = farms
fallowed during the outbreak; = ISA positive farms; = ISA positive farms
fallowed; = populated farms containing non-susceptible species (halibut, ballan
and goldsinny wrasse); = fallowed non-susceptible species farm (halibut) & =
processing plant. (Two fallow farms included in Figure 2 are located off the foot of
this map).
Five sites (g-k) were harvested during January 2009 including the officially suspect
site, g, which had by then been confirmed to be ISAV negative after a 150 fish test.
All of these sites had tested negative, most at the 150 fish level, although site h was
only subject to a smaller diagnostic test, prior to it being fallowed. Thus half of the
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salmon sites in MA 3a that had been populated in December 2008 were empty by the
end of January 2009 (Figure 3.II), as a result of normal planned harvesting activity.
There were diagnostic indications of ISAV from two sites in January 2009, however,
these were not confirmed. One site (D) tested positive in 2 of 3 replicates from one
pool when tested by qPCR. This, as described in the methods, was officially a
negative result however it might indicate exposure to infection. Five months later on
14 May 2009 positive tests were obtained from this site which became the fourth to
be confirmed. Site F tested positive by qPCR on the 7 January 2009, however, it
was not confirmed by virology. A subsequent 150 fish test from this site on 13
January was negative by both qPCR and virology. However, the site was confirmed
as ISAV positive nearly 9 months later on 30 October 2009 during harvesting. Even
if these sites had been positive, due to them being located in MA 3a, they were in
any case under movement restrictions and enhanced surveillance. As such, their
treatment as negative in the absence of clinical ISA was considered to be of low risk,
although their subsequent histories suggest sites D and F may have been carrying
infection for a long time before they were confirmed ISAV positive.
During January 2009 site B tested positive by qPCR (4/30) and virology (8/30) to
become the second confirmed case of ISA on 30th January 2009. This site was
cleared of fish by the 6 March. Another ISAV negative site, l, was harvested on 25
February. The trout producing site m was culled on 2 April for welfare reasons,
although the fish tested ISA negative. The site was located in shallow water so as
water temperatures increased and the fish grew larger their stress was increased.
Traditionally these trout were moved to a site in a different MA but this was prohibited
by ISA movement restrictions. By 7 March 2009, within MA 3a, there were 7 stocked
sites: 4 salmon farms; the sea trout site and the 2 non-susceptible species sites
(Figure 3.III).
Site C, adjacent to positive site B, tested positive from diagnostic samples taken on
11th March 2009. This became the third confirmed ISA positive site following a 150
fish test on the 20th March (16/30 by qPCR and 15/30 by virology). The site was
fallow by the 8th April. In April, the halibut site n was harvested, but these fish are
non-susceptible so this is of no relevance to ISA control (apart from increased boat
traffic).
A fourth site D was confirmed ISA positive on the 18 May 2009 from samples
collected on the 14 May. This site was rapidly confirmed on the basis of not only
qPCR (1/1) and IFAT (1/1) testing but also histological signs of ISA and increased
mortality. However, this site was slow to fallow and was cleared of fish by 2 July.
A fifth site E was confirmed at nearly the same time (21 May) on the basis of samples
taken on 19 May (25/30 qPCR; 26/30 virology and 12/12 by histology). The site had
16
been sampled after a veterinarian reported signs consistent with ISA. This was a
small research site and was fallowed by 27 May (Figure 3.IV).
By the second week of July 2009 only site F remained stocked with susceptible fish
in MA 3a as there was insufficient evidence, from monthly inspection at that time to
confirm the presence of the disease. This site was close to sites from which infection
had been reported in spite of an early positive qPCR result (7 January 2009), signs of
ISA were not observed until October 2009. Visiting FHIs observed gross pathology,
prompting the sampling of moribund fish, leading to confirmation of ISAV. The site
was confirmed positive on 30 October on the basis of cell culture, qPCR and clinical
signs. As no other sites containing susceptible species were populated for 4 months
prior to this it is likely infection persisted undetected on F for at least this time period.
Site o was a very small (Table 2) tank-based research site containing non-
susceptible species. This was not considered to be a risk for ISA and so was not
depopulated.
3.1.1. Diagnostic Testing Outside MA 3a
All the sites located out with MA 3a that were tested gave negative diagnostic results
for ISAV. The complete list of diagnostic test results from within Shetland but outside
MA 3a are presented in Table 2 of Appendix 2. Most fish were tested by qPCR and
virology, but some by qPCR only, 1352 samples were taken from 685 fish. A total of
15 sites were sampled by qPCR and 12 of these were also tested by virus isolation.
Sampling was concentrated on high risk sites with 600 fish taken from 4 sites that
might have been exposed directly or indirectly by fish movement: two in MA 4a, one
in MA 3b and one in MA 2b, this last being the site nearest to LPP (Figure 6).
3.1.2. Clinical ISA
The positive sites were confirmed on the basis of qPCR and virology testing,
although pathology allowed a more rapid confirmation of sites, including site D,
before the virology results (which later supported the confirmation) were available.
During the outbreak, evidence of both infection and clinical disease were found in MA
3a. Pathological changes associated with ISA; observations from fish sampled at
each confirmed site are illustrated in (Table 3). Gross pathology is determined by
OIE guidelines and a clinical score sheet used to assess liver colour score.
Experimentally challenged salmon using virus obtained from site B developed
diagnostic features of clinical ISA and suffered elevated mortality, although mortality
was slower to occur than from the 1998-390 strain (K. Urquhart in prep.). The 2008-
0982 ISAV was undoubtedly the causative agent of disease.
17
Table 3
Diagnostic features associated with clinical ISA found in fish from confirmed sites A-
F. Liver colour is on a scale where values 8 raise suspicion of ISA.
Site Pale Gills Zoned Gills Pale Heart Dark Liver Bloody
Ascites
A Y Y
B Y Y 10 Y
C Y Y 9 Y
D Y Y 8/9
E Y Y Y 9/10 Y
F Y Y 8/9 Y
The confirmed sites C, D and E all exhibited mortality rates above 1% week-1 over
several weeks in the worst affected cages. Mortality at sites B and F was generally
low only reaching 1% week-1 on one occasion at each site. Mortalities >0.05% week-
1 were widespread across most cages on affected sites, except F. At confirmation,
site A was fallow, however mortality rate was reportedly as high as 7% per week, on
the worst affected cage at the time of the index case. Elevated mortality was
attributed by the site operators to causes other than ISA and certainly other
pathogens were reported as present and confirmed by laboratory diagnosticians.
The fish on all sites exhibited gross pathology, lethargy and morbidity, prompting
diagnostic investigations by the FHI, and following laboratory analyses to determine
their cause.
3.2. Potential Origins of the Outbreak
Three possible origins of the ISAV affecting farmed Atlantic salmon in south west
Shetland were considered. (1) It may have evolved spontaneously from an avirulent
wild type virus such as HPR0 (Nylund et al. 2003), (2) it could have been introduced
with imports from abroad, or (3) the 1998/9 virus might have persisted undetected.
Evolution of virulence in ISAV is considered to involve a simple deletion mutation in
the hypervariable region (Krossoy et al. 2001; Nylund et al. 2003). Although
individual sites were fallowed following the 1998/9 outbreak, MA 3a was not
synchronously fallowed and therefore, at the MA level, was considered to be
continuously stocked. This could allow virus to circulate over many generations,
facilitating the emergence of virulent strains (Murray and Peeler 2005). Other
pathogens including salmonid alphavirus (SAV), infectious pancreatic necrosis virus
(IPNV), Renibacterium salmoninarum (the agent of bacterial kidney disease, BKD),
and sea lice, had built up in MA 3a. These intercurrent infections would stress the
fish potentially making them more susceptible to ISA infection. The MA is treated as
6 regions in the Code of Good Practice for Finfish Aquaculture (CoGPWG 2006) and
18
use of such small areas for management may be inappropriate for prevention of ISA
emergence (Beattie et al. 2005).
ISAV could have been introduced with imported batches of ova or movement of
equipment. Clinical ISA has been controlled in the Faeroes at great cost and in the
initial absence of good biosecure practices, but continues to be present in Canada
and Norway. Scottish contacts with Norway are much greater than with Canada.
There is no evidence of foreign imports of live fish or dead fish for processing in the
SPP and therefore there is no evidence MA 3a could have been exposed to ISAV via
this route. Vessels servicing farms are locally based (see later section) so would not
have contact with Norwegian fish. Norwegian ova were used to produce some of the
smolts that were stocked to the index site A. However, the epidemiology of ISA (not
affecting sites that received fish from the index site) indicates virulent ISA likely
emerged late in the fish production cycle and if so, virulent ISAV was not considered
to be introduced with the ova. In 2007, 33 million Norwegian ova were supplied to 16
Scottish mainland and Western Isles hatchery sites, and these were subsequently
distributed to numerous sea sites within Scottish waters, with no evidence of ISA
apart from the Shetland outbreak.
It is possible that HPR0 was present in ova or fish products imported from Norway, or
elsewhere, and careful consideration should be given to its evolution (Kibenge et al.
2009). Since HPR0 is already present in Scotland, with no association with clinical
disease, (McBeath et al. 2009), in this case, such imports are currently considered as
representing a relatively low risk of emergence. The risk would increase if HPR0 was
common in imports relative to its indigenous prevalence and resultant increased
prevalence of HPR0 increased the chance of an emergence. This is quite different
from a country in which HPR0 and virulent ISAV were both absent (Kibenge et al.
2009).
It is improbable that the virus that caused the 1998/9 outbreaks could have persisted
unnoticed for 10 years. The 1998/9 ISAV (1998-390) caused high levels of mortality
so would rapidly have emerged as a problem on affected farms. It is unlikely such a
problem would not have been noticed, although restricted outbreaks could have been
misdiagnosed. Genetic evidence shows the virus at 2008-0982 was a European
genogroup 1 isolate, while the 1998-390 virus belonged to genogroup 3 (Nylund et al.
2007) and mortality took longer to occur in fish experimentally challenged with the
2008-0982 virus relative to those challenged with the 1998-390 virus (K. Urquhart in
prep.).
The source of the virus behind the outbreak remains unknown however it is valuable
to consider potential sources of introduction, as an aid to the control of future
outbreaks.
19
3.2.1. Genetic Epidemiology
A comparison of the haemagglutinin-esterase protein gene sequences of 2008-0982
and other EU-G1 isolates has been undertaken (Figure 4).
The analysis appears to cluster 2008-0982 with three EU-G1 isolates from Norway
(AY973179, FM203284 and FM203285). However, this is poorly supported with a
bootstrap value of only 53% and the analysis does not therefore identify the 2008-
0982 isolate as being an inclusive member of any particular terminal subgroup.
Previously obtained isolates similar to 2008-0982 have been identified from several
geographical areas along the Norwegian coastline rather than a single locality.
Closely related non-virulent strains of the virus (HPR0) also occur and the possibility
that one of these has recently evolved a capability to cause disease cannot be
discounted. We conclude that, while the sequence data are not inconsistent with a
Norwegian origin of 2008-0982, they do not provide definitive evidence of this.
Figure 4 A maximum likelihood tree of 2008-0982 and other EU-G1 isolates.
The genetic evidence thus rules out that the ISAV was the same as the 1998-390
strain that caused the 1998/9 epidemic, but does not distinguish between a
Norwegian or local Scottish origin.
3.3. Spread of the Outbreak
3.3.1. Movement of Live Fish
Movement of live fish from infected sites is a high risk activity for spreading disease,
in the absence of good biosecure practices. The movements of fish within, into and
out of, MA 3a have been used to develop a contact structure network (Munro and
Gregory 2009) (Figure 5). This figure illustrates that there is no evidence to link ISA
positive sites with the movement of live fish. The history of individual fish movements
has not therefore been tracked in detail. However, there were substantial numbers of
movements across MA 3a’s boundaries.
Other EU-G1
2008/0982
FM203285
AY973179
FM203284
8
53
20
Two movements of live fish occurred from the putative index site A and the two sites
that received them were immediately put under official suspicion of being infected.
Fish from one of these sites had already been harvested and processed therefore
samples were not obtained (and the site is therefore not listed in Table 2). However,
fish from the other (site g) were tested and found to be negative for ISAV. Since the
movement of fish is high risk, virulent ISAV is likely to have been absent or, if
present, at low levels at the time of the movement on 27 June 2008.
It is possible that if stocks on putative index site A were kept separate then only
uninfected stocks might have been moved off. This would still argue for very low
prevalence in the infected stocks at the time of movement, itself suggesting infection
could at the earliest have occurred only shortly before the June 2008 movement.
There were no movements of live fish from any sites in MA 3a after 27 June 2008,
with the exception of transport to SPP and LPP (detailed in section 3.3.2.c, including
‘bus stop’ harvests). There were, however, 12 seawater to seawater movements
within MA 3a before 27 June 2008. There were also movements to and from sites in
other MAs. Live fish were moved into MAs 4a and 2b and from MAs 4a, 3b, and 2c
(Figures 5 & 6). If ISAV was not present before June 2008 these fish movements
could not have spread the virus, however the sites out with MA 3a were visited by
FHI to confirm the absence of ISAV, suggesting it was not present in this MA before
June 2008.
21
Figure 5 Structure of the network for movements involving salmon and trout in MA
3a. Circles are proportional to numbers of other sites contacted. Letters are site
listed in Table 2; unlabelled nodes are sites not listed in the table (either harvested
before site A was confirmed or in other MAs).
One movement occurred on 6 October from MA 3b to a site in MA 3a, and by this
time ISA might have been present. This movement was from outside the affected
MA, and therefore a far lower risk than a movement going the other way. A risk may
still have existed of spreading infection with empty equipment moving to collect these
fish (Peeler and Thrush 2009) but there was no evidence of transmission of infection
to the source site.
= Marine site
= Freshwater site
= processing plant
Black = ISA +ve
White= ISA –ve
Grey = untested
MA 3b
E MA 2c
D
F
MA 4a
k
j
h m
g
SPP
A
MA 4a CMA 2b
Sites circled in
black a e outside r
MA 3a
LPP
B
il
22
Figure 6 Locations of MAs in risk contact with MA 3a through movement of live fish
or ‘bus-stop’ harvests to processing plants. = live fish moved to 3a; = live fish
moved from 3a; = bus stop to and from 3a; = bus stop to 3a; = fish farm and
= processing plant. The shaded grey areas represent an amalgamation of tidal
excursions around the fish farms.
All seawater to seawater movements are high risk in relation to the risk of disease
spread and subject to assessment under the industry code of practice (CoGPWG
2006). Had the outbreak begun earlier than it appears it did and assessments did
not consider the presence of ISA in the area (if it had not yet been confirmed),
23
infection could have been more widely spread (Figure 6). However, movements
ceased in the last 6 months of 2008, before ISA would appear to have originated and
there is no association between ISA outbreaks and the movement network (Figure
5). None of the 15 sites outside MA 3a that were tested returned ISA diagnostic
positive results.
3.3.2. Harvest Operations
3.3.2.a. Processing Plants
Salmon from sites in MA 3a were primary processed at either SPP within the area,
and LPP in Lerwick on the east coast of Shetland. Both plants have advanced
disinfection regimes and although it has not been independently confirmed that they
were fully functional at all times Marine Scotland has found no evidence to the
contrary, following risk assessments of the movement of live and dead fish to harvest
(Fraser and Murray 2009). If disinfection was fully functioning then the associated
risk of ISAV spreading was negligible according to the analysis of Jarp and Karlsen
(1997), although the risk would have been substantially enhanced if effluent
disinfection was not employed.
The LPP did not receive fish from MA 3a between May and 11 December 2008, so it
could only have been potentially exposed to infected material from December
onwards. With notification of the presence of ISA on 2 January 2009 in MA 3a staff
at LPP would have been aware of the risk of infection and biosecurity would have
been an even higher priority.
This plant has facilities to hold live fish in adjacent marine cages. This is considered
a high risk (Munro et al. 2003) as, due to their immediate proximity, such fish could
potentially pick up infection released from the plant. Fish might then act as a
continuous source of infection to visiting vessels, level of risk could increase with
time on site. A similar situation is believed to have played a major role in the spread
of ISA in 1998/9 (Murray et al. 2002).
The only site within 5 km of LPP was tested at the 150 fish level and placed under bi-
monthly surveillance due to the potential risk should disinfection have failed (Jarp
and Karlsen 1997). However, this site showed no evidence of infection. All sites
near the SPP which might have been at risk had disinfection failed, were already
under enhanced surveillance as they were within MA 3a.
3.3.2.b. Transport Vessels
The transfer of fish to processing plants represents a series of risks for transmission
of infection, particularly to other MAs (Munro et al. 2003; Fraser and Murray 2009).
These risks can be minimised by processing within the MA (at SPP), however this is
24
not always practicable and fish have been, and are being, transported to LPP for
processing. Two alternative practices were risk assessed. (1) Dead fish could be
transported in boats to the Scalloway pier and then moved overland to Lerwick where
they are unloaded into the LPP processing plant. Alternatively (2) fish could be
shipped alive to LPP and pumped directly into the plant for processing.
The relative risks of these two processes have been reviewed using methods
developed by Munro et al. (2003) and Fraser and Murray (2009). The greatest
apparent risk from shipping live fish is transport out of the MA, however so long as
the wells containing the fish on the vessels remain sealed and the vessels undergo
disinfection (CoGPWG 2006), these fish represent little risk to the environment.
Risks associated with the alternative on-site slaughter and movement of dead fish to
LPP present several opportunities for infection to spread (at slaughter, at the
Scalloway pier when unloaded, and at LPP when transferred). It is therefore
probable that the risks associated with movement using well-boats is not greater, and
may be lesser, than those associated with on site slaughter and overland transport.
Each of the processing plants had one dedicated vessel which serviced its own
farms, except for a brief period in August 2008 (8-27) when a replacement vessel
was used for LPP. This was before LPP had processed material from the control
zone and so was of negligible risk. This made it relatively straightforward for Marine
Scotland scientists to obtain copies of the vessel logs to undertake an analysis of
movement patterns.
The SPP received fish from only one source outside Shetland, this being in Orkney.
The fish were received from March to June 2008, likely before infection became
established at index site A, and so these movements are very unlikely to have
represented a risk, to be certain the Orkney site was inspected by the FHI. Harvests
were taken from sites around Shetland, including the islands of Unst and Yell (Figure
6), to SPP. Most fish were entirely processed here, but some did go to packing
stations prior to processing at SPP.
The spatial pattern of outbreaks, concentrated in a relatively small area close to the
putative index site, is not consistent with well boats as a major vector of transmission.
If it were, this would have resulted in cases throughout Shetland, reflecting the
movements of these vessels. This different pattern of scattered cases of ISA
infection, caused by well boat movements did occur in 1998 (Murray et al, 2002).
3.3.2. ‘Bus Stop’ Well Boat Movements
The vessel servicing SPP engaged in repeated use of ‘bus stop’ harvests, picking up
a partial load of fish from one site then going to another to complete a full load en
route to a processing plant. If the first site is infected, then infection might be
transmitted to the second by this movement. These ‘bus stop’ harvests occurred on
25
at least 71 occasions in 2008 with some of these involving movements between MA
3a and other MAs (Figure 6). Seven ‘bus stop’ movements involved the putative
index site A, these occurred during the September to November 2008 period when
ISA may have been present (Figure 7). Movements in which the index case was the
second site to be visited were of low risk, but those where it was visited first
represented higher potential risk. One such visit involved a site in another MA, (MA
3b) (Figure 7). The level of risk depended on levels of biosecurity practiced by the
operators of the well boats and time that fish remained on site after the visits. Due to
these ‘bus stop’ activities being involved in harvesting operations, the sites may be
quickly cleared and this is quite different from ‘bus stop’ deliveries involving smolts.
There were a further 5 ‘bus stop’ harvests from MA 3a to 3b and 7 from MA 3b to 3a,
excluding the harvests involving the putative index site (Figure 6). There were 2
harvests from MA 4a (North West Shetland) to 3a and also 2 from MA 2a (north east
Shetland) to 3a. All these ‘bus stops’ involved sites around the mainland island of
Shetland, but MA 2a is on the east coast, so there is a potential for ‘bus stop’
activities to result in long distance transmission. In this instance the movement was
into, not out of, MA 3a so the risk of spread was lower.
26
Figure 7 ‘Bus stop’ harvests involving the index site A. = fish farms; =
processing plants; solid lines between fish farms represent ‘bus stop’ harvests;
numbers refer to the number of ‘bus stops’ and the dashed line represents
movements to the processing plant. In the 4 harvests involving site k, salmon were
taken from here before the well boat visited the index site A. However, when fish
were taken from the other 3 sites, including a site in MA 3b, the well boat had visited
index site A first.
The other vessel’s log is less clear so we do not know if such activities were the
normal practice. Only sites visited are logged (sometimes only the company name
without a specific site location was recorded) and not whether the vessel returned to
the processing plant between the site visits. However, this vessel was not active in
the area for most of the period before ISAV was detected.
There is no evidence that infection was spread by the movement of these vessels out
with the controlled MA, although this was clearly the case in 1998/9 (Murray et al.
2002). Improved disinfection at processing plants and for vessels may have been
important in this. However, the practice of ‘bus-stop’ deliveries remains a risk and is
contrary to the CoGP which stipulates stage 2 disinfection when well boats leave an
existing site to start work at another site (CoGPWG 2006).
27
3.3.3. Vertical Transmission
3.3.3.a. Movement of Smolts
Some of the fish on the index site A were reared from Norwegian ova. These could
be the source of infection, however given that ISA seems to have appeared on site
late in the production cycle it is unlikely that infection was within the ova via vertical
transmission. In 2007, 33 million Norwegian ova were supplied to Scotland with no
evidence of ISA, apart from the Shetland outbreak.
Smolts were imported into MA 3a from various producers around the UK (Shetland,
the Western Isles, mainland Scotland and England (Figure 8). Essentially ISA is a
marine salmonid disease with almost all reported cases being from marine salmon,
and those from freshwater involve some contact with sea water (Jarp and Karlsen
1997). Due to this marine nature, and there was no reason to suspect ISA in the UK,
these freshwater sources are very unlikely to represent risks of ISAV infection to
Shetland.
The pattern of contacts in Figure 8 represent a network by which ISAV could
theoretically have spread throughout the UK, as pathogens might be transported with
equipment going to the smolt producers (Peeler and Thrush 2009). However, the
risk appears negligible because the overall risk consists of four very low risk steps
that combine to produce this negligible risk: (1) Only empty transporters moved from
MA 3a to the remote sites, so risk of transfer to these sites is extremely low; (2).
These sites are freshwater, while ISA is essentially a marine disease, except
occasionally in broodstock recently moved to freshwater (Lyngstad et al. 2008).
Therefore infection is unlikely to spread even if ISAV reached the smolt producers;
(3) Sometimes there was no direct contact between the sites and well-boats, with
smolts being moved to the well-boats overland, so contact between MA 3a and the
smolt producers was often broken; (4) The freshwater to seawater movements into
MA 3a all occurred before June 2008 and therefore before infection was established
at the putative index site A.
28
Figure 8 Sources of smolts moved into MA 3a within the year 2008. = source fish
farms.
Previously ISAV has been found in wild freshwater salmonids using PCR (Raynard et
al. 2001) however the spread appears limited. The widespread sourcing of smolts is
probably of more concern for the spread of diseases such as BKD or infectious
pancreatic necrosis (IPN) that exist in both marine and freshwater stock (Murray
2006). Such diseases may have the potential to spread around the UK, and as such
they should be monitored.
29
3.3.3.b. Vertical Transmission and Movements of Ova from Shetland
There is inconclusive evidence for vertical transmission of ISAV (Lyngstad et al.
2008; Vike et al. 2009). If it could occur via the movements of ova, there is the
potential risk of the spread of ISAV within Scotland and beyond via Atlantic salmon
broodstock reared in Shetland. The only broodstock site on Shetland is in MA 1b on
the island of Unst (Figure 6).
There were no movements of live fish out of the control and surveillance zones to
sites outside mainland Shetland, and none at all out of MA 3a after June 2008. As
such it is reasonable to conclude that there was negligible risk of transmission to
sites on Unst as a result of live fish movement.
Harvest vessels from SPP visited sites belonging to the company that owned the
broodstock site in July, August and September 2008, times when the plant could
potentially have been processing infected material from MA 3a. It is therefore
possible that there may have been contacts with sites within the MA from which the
broodstock originated. This risk is probably negligible if disinfection at the SPP was
correctly implemented.
The evidence for vertical transmission of ISAV is ambiguous (Lyngstad et al. 2008;
Vike et al. 2009) and the broodstock fish exhibited no signs of excessive mortality
when visited by the FHI. The eggs were disinfected on 3 occasions: immediately
following stripping when “soft”; before transferring and at the receiving site in
mainland Scotland. In these circumstances, and given the extremely low risk that the
broodstock were exposed in the first place, the overall risk of vertical transmission
must be regarded as negligible (Peeler et al. 2007) in the presence of good
biosecurity practiced on all occasions.
If evidence showed that the risks posed by these broodstock were considered to be
non-negligible, the same risk would apply to all broodstock sites sharing a MA with a
site that had used a processing plant that had processed fish from any MA in which
ISA was present. This is a stricter definition of risk than is imposed on imports from
Norway, a country where ISA is endemic. It would be contrary to current evidence to
impose such a restriction on Scottish smolts without equally imposing it on imports.
The investigation has shown no association with Shetland broodstock/ova and the
Shetland Islands ISA outbreak.
3.4. Wild fish
As part of the epidemiological survey wild fish were screened for ISAV using qPCR
(Wallace et al. 2009). Two discreet field sampling trips were undertaken by Marine
Scotland staff, the first for marine and, the other for fish caught in freshwater with the
30
specific aim of sampling salmonids. Saithe from within an infected salmon farm were
also sampled opportunistically.
A total of 1196 wild marine fish from 12 species were caught from the 3 trawl areas
within MA 3a (Figure 2 and Table 4). Area (1) was in the vicinity of the then actively
infected site B making this a good time to sample wild marine fish and 446 from this
area were sampled. The kidney and gill samples were thought most likely to carry
ISAV so these were initially screened. All these samples tested negative for ISAV by
qPCR and for this reason the brain and heart tissue were not screened.
Table 4
Wild marine fish sampled for ISAV in February 2009. Numbers refer to the trawl
locations (Figure 2).
Species Papa –
Hildasay (1)
North Havra
– Fora Ness
(2)
Scalloway
Deeps (3)
Long rough dab (Hippoglossoides
platessoides) 150 50
Saithe (Pollachius virens) 30 10 30
Whiting (Merlangius merlangus) 35 150 110
Common dab (Limanda limanda) 100 150
Norway pout (Trisopterus esmarki) 150
Herring (Clupea harengus) 30
Plaice (Pleuronectes platessa) 55
Short-horn sculpin (Myoxocephalus
scorpius) 70
Atlantic cod (Gadus morhua) 4 25
Flounder (Platichthys flesus) 2
Haddock (Melanogrammus
aeglefinus) 30
Lesser Argentine (Argentina
sphyraena) 15
Total 446 540 210
Results from the freshwater sampling are presented in Table 5; refer also to Figure 2.
In total, 216 fish caught from the freshwater environments were sampled comprising
178 salmonids and 38 non-salmonids. The later sampling date was chosen to allow
the freshwater temperatures to warm up. From previous personal experience these
higher temperatures make the fish more active thus increasing the chances of
catching them. Additionally there would be a greater chance of catching migratory
sea trout returning to freshwater at this time of year.
31
Table 5
Sampling of fish caught in freshwater for ISAV in March 2009. Numbers refer to site
locations (Figure 2).
Species
Stromfirth
burn 23rd
March (4)
Stromfirth burn
24th March (4)
Weisdale
burn (5)
Scalloway
burn (6)
Atlantic salmon parr
(Salmo salar) 30 34
Brown trout (Salmo
trutta) 2 1
Sea trout (Salmo
trutta) 5 (3 escapes) 2
Brown/sea trout 22 82
European eel
(Anguilla anguilla) 22 3 8 1
3-spined-
stickleback
(Gasterosteus
aculeatus)
2 1
Flounder
(Platichthys flesus) 1
Total 62 40 30 84
This sampling also coincided with an active infection in site C, which was in the
process of being harvested. Kidney and gill samples from all these fish were
screened using qPCR and found to be negative. Wild salmonids are particularly
likely to carry ISAV infection (Nylund and Jakobsen 1995; Raynard et al. 2001) so
these negative results are of particular importance.
Two saithe taken from within an infected cage at the third ISA confirmed site (C)
were individually sampled. Gill and kidney samples were screened for ISAV using
qPCR with both gill samples testing positive (2/2). This small sample size indicates a
low population of saithe in the infected cage however those that were present had
been exposed to infection. Saithe are resistant to ISAV infection and quickly clear
the virus (Snow et al. 2002; McClure et al. 2004a) so they are unlikely to be effective
vectors. The qPCR results probably reflect exposure of the gills to virus laden water
rather than active infection.
With the exception of the saithe taken from within a clinically infected farm wild fish,
even salmonids, showed no sign of infection with ISAV. We conclude that wild fish
were of low, if any, importance in the initial spread of the virus. This does not rule out
the possibility that prolonged exposure would result in their becoming infected,
however there has been, to date, no evidence of mortality or clinical ISA in wild fish
populations from any ISAV infected country.
32
3.4.1 Seal attack and Associated Activity
Mortality records collected from ISA affected farms later in the outbreak attributed
very high levels of mortality to seal attacks. This self reporting is not independently
confirmed, but high levels of attacks on sick fish could allow for escapes to occur and
potentially the spread of infection. Such fish can travel large distances and collect
near farms where they can obtain waste feed (Dempster et al. 2009; Uglem et al.
2009). Grey seals (Halichoerus grypus) are known to travel long distances at 75-100
km d-1 (McConnell et al. 1999), but the virus is destroyed within the digestive system,
and other associated risks are deemed low.
3.5. Hydrodynamics and Spread
Proximity to an infected site could be a risk factor for a variety of reasons, but
transmission through the water is strongly indicated. Movement through the water is
the only explanation for the downstream affect found by Gustafson et al. (2007), and
shared contacts are explicitly included by shared ownership in the model of Scheel et
al. (2007). Hydrodynamic spread is believed to be important in the epidemic
currently affecting Chile (Mardones et al. 2009).
As a result of this hydrodynamic transport one of the principle controls on ISA in
Scotland is based on tidal excursion modelling and the existing MA 3a was defined
by this model. The model was developed during the ISA outbreak in 1998/9 and
uses simple oceanographic assumptions to define a distance over which ISAV could
be moved by tidal currents (Stagg 2003). The distance is 3.6 km in Shetland (the
distance is twice this on mainland Scotland where tidal ranges are greater). Sites
falling within this distance are included within the control zone, while a surveillance
zone is set up for all sites around the control zone until a separation distance is
reached where tidal excursion zones do not overlap. This is an effective way of
deriving zones on which controls can be imposed.
33
Figure 9 Map of sites involved in the outbreak. The letters refer to the farms listed in
Table 1: = farms depopulated prior to the outbreak; = ISAV positive; = ISAV
negative; = non-susceptible species; = processing plant. Sites B, C and D are
within 3.6 km of site A; E and F are slightly outside this but well within overlapping
tidal excursion distances.
Simple modelling indicated ISAV was unlikely to be transported beyond the tidal
excursion zone due to its rapid decay, meaning longer-term hydrodynamic
movements were unlikely to be relevant (Murray et al. 2005). All affected sites, apart
from the putative index case, were within overlapping tidal excursion distance of an
infected site and most were within a single radius of an infected site (Figure 9). The
sites E and F are slightly more than 1 tidal excursion distance, but within overlapping
excursion distances: this overlapping distance is the standard model used to identify
sites at risk in both this and the 1998/9 outbreak. Both sites are within the excursion
radii of sites within 1 radius of A, and so infection may well have spread indirectly.
34
Site F is connected by exposed open sea to site A and therefore could have been
exposed to virus transported via a short-term current. Infection was very slow to
develop at site F, which might indicate exposure to a very low dose of virus from A.
Site E is within a tidal excursion of SPP, so had there been a failure of disinfection it
is possible that this could, also via hydrodynamic transport, be the source of
infection. Although prevailing offshore currents may be stronger than coastal
currents, the significant separation distance between MA 3a and its neighbours
means water-borne transmission to other MAs is unlikely.
Local intra-company contacts might explain the local spread and shared ownership is
a risk factor for ISA in Norway (Scheel et al. 2007). In Shetland ISAV has been
detected in sites belonging to 4 separate companies (Table 2) and there was no
movement of fish between affected sites (Figure 5). Sites B, C and D belonged to
one company, so intra-company contacts to B and/or C is a possible explanation of
spread. The FHI found these sites (B, C and D) had site-specific equipment which
was not moved off site and site dedicated boats. Furthermore any such intra-
company transmission would apply to cases that occurred months after the putative
index case had been confirmed and so biosecurity should have been a high priority.
No transmission occurred to the other sites belonging to the company that owned site
A, even although ISAV had not been detected, and heightened biosecurity was not in
place.
The highly clustered nature of the outbreak, all cases within a few km of the putative
index case argues for environmental transmission. The pattern does not fit with
transport of fish, harvest vessel movements or company ownership. It is possible
that wild fish could have spread infection, but in the absence of positive results
(Wallace et al. 2009) and the established association of ISAV transmission with water
movements, hydrodynamic transmission of infection is the most likely driver of this
outbreak’s spread.
3.6. Limitation of the Outbreak
The 2008/9 ISA outbreak appears to have been localised to a relatively small area,
although it spread widely within that area. This is unique, with other outbreaks
including the 1998/9 Scottish outbreak becoming widespread rapidly (Murray et al.
2002). Similarly outbreaks in Chile spread from region X to regions XI and XII and
multiple clusters of local spread developed (Mardones et al. 2009). In Norway a
variety of different ISAV genogroups were found scattered along the coast, arguing
that long-distance spread events are fairly frequent (Lyngstad et al. 2008). The ISAV
has also spread between the different salmon farming islands in the Faeroes
archipelago. While infection demonstrates a pattern of transmitting most easily in
clusters, with relative risk about 10 times greater (Mardones et al. 2009), the virus
frequently is transmitted over long distances to form new clusters or isolated
outbreaks.
35
The spread of the 2008/9 ISA outbreak appears to have been dominated by water
movement. This is in contradiction to the 1998/9 outbreak, for which vessels and
movement of fish between different areas of the country spread ISA to virtually all
areas of Scotland in which salmon were farmed (Murray et al. 2002). A variety of
routes of spread (seawater-to-seawater movement of live fish, harvest operations
and wild fish movement) were identified, but none were associated with the spread of
ISAV. It appears that strategies developed as a result of the 1998/9 outbreak (Anon
2000; CoGPWG 2006) had been effectively applied. This also corresponds to results
from Norway where Aldrin et al. (2010) found shared ownership was a small risk
factor compared to close neighbours. However, practices were identified which had
the potential to increase the spread of infection; this underlines the need for industry
to be vigilant in the application of good biosecurity practices.
The seawater-to-seawater movement of fish did not occur in late 2008, but such
movements had occurred earlier, so had the outbreak occurred earlier it could have
been more widely spread. Harvest vessels that played an important role in the
1998/9 spread may have avoided contamination, because effective disinfection
appears to have been in place at the processing plants that received fish from sites in
the MA. However, inappropriate practices of bus-stop harvesting were occurring
frequently and so the risk of transmission by well boats did exist, even between MAs,
involving risks in the absence of good biosecurity (Fraser and Murray 2009).
Wild and escaped fish caught early in the outbreak were found to be negative for
ISAV (Wallace et al. 2009). As wild fish are established vectors of ISAV (Nylund and
Jakobsen 1995; Raynard et al. 2001), the patterns of movement of fish between sites
(Uglem et al. 2009) and the exposure of wild fish to viruses in the vicinity of infected
farms, as illustrated by IPNV (Wallace et al. 2008) there is the potential for wild fish to
transmit infection, where it remains on farm sites.
Smolts were supplied into the MA area at the Great Britain level; this was not
associated with spread of ISAV and probably represents a greater risk of spreading
diseases with a freshwater component, such as IPN (Murray 2006). However in
1998/9 marine site-to-site movements occurred over 100s of km and contributed to
spread of ISAV at the Scotland scale (Murray et al. 2002), these movements
continue, but at the regional scale (Figure 6). Ova are imported from several
countries, including Norway, and so although we have no evidence of this having
occurred in the UK the potential for vertical transmission should continue to be
investigated (vertical transmission in Chile is currently suspected (Vike et al. 2009)).
Blue mussels (Mytilus edulis) are abundant in Scottish waters and these adsorb and
inactivated ISAV rapidly, so are not likely to act as reservoirs (Skar and Mortsen
2007). Sea lice may potentially act as vectors of infection (OIE 2009). Lice from a
treated or culled site might drop off their hosts and be transported to neighbouring
farms in large numbers so may enhance spread at the local level.
36
Other routes of spread may also exist at the regional level, particularly in the absence
of good biosecurity, so long as there is a source of virus. These may include other
vessels such as ferries or fishing boats, harvest and grading equipment, divers or
other staff visiting sites, birds or other predators. Even hydrodynamic spread
between MAs is possible, since current velocities can be large. Since a MA cannot
be sealed off, the possibility of spread to other MAs can be reduced but cannot be
eliminated so long as infection (and especially disease) is present.
3.6.1. Removal Policy
Rapid removal of salmon from confirmed infected sites and the implementation of
good biosecurity practices were factors believed to have played a critical role in
control of the 1998/9 outbreak (Stagg 2003; Mardones et al. 2009). Aldrin et al.
(2010) found a history of infection was not a risk factor for ISA in Norway, so
depopulation is effective at removing the virus. Other countries have allowed
delayed removal or require mandatory removal of fish only after mortality reaches a
trigger level. In Scotland site-level depopulation is carried out, even if ISA is
restricted to a few cages, but in other countries depopulation may be applied only to
cages with clinical disease. McClure et al. (2004b) showed that the prevalence of
ISAV in non-moribund fish was the same irrespective of whether or not mortality
occurred in a cage provided there was mortality on the farm; this observation
supports site-level culling.
In the Shetland 2008/9 outbreak depopulation was carried out within 7 weeks of
confirmation. The putative index case site A had already been harvested for 13 days
at the time of confirmation. The other cases were harvested 35 (site B), 19 (site C),
45 (site D), 6 (site E) and 26 (site F) days respectively after confirmation (mean 26
days = 3.7 weeks). This may be compared with a mean of 21 weeks in Chile
(Mardones et al. 2009), where ISA became widespread within the industry.
However, 3.7 weeks is marginally longer than the 3 weeks removal time achieved in
1998/9 (Stagg 2003). Identification of a practical rapid and biosecure method of
removal delayed the clearance of fish from site B. However, once a suitable
method was identified, the site was depopulated in a 9 day period. The sites C
and E were rapidly depopulated; site E being a small experimental facility.
Depopulation of site D was slower, by this time only 2 other sites remained populated
with susceptible species (salmon) within the MA 3a, and one of these was already
infected so potential for local spread was limited. However, MAs can never be 100%
isolated and so depopulation was eventually achieved 6.4 weeks after confirmation.
The increased shedding from clinically affected fish on confirmed sites increases the
risk of horizontal transmission and the possibility for spread, even between MAs.
This would increase the longer fish are left on site. The risk is more than linearly
increased with time because farm-level prevalence of infection increases and clinical
disease is increasingly likely. The potential for escapes in infected populations,
37
especially after seal attacks, increases the risk of fish movement between MAs. The
consequences of infection escaping the MA are very large and therefore the
principles of risk analysis (non-negligible probability × high consequence) indicate
that leaving infected fish on site is a serious risk (Peeler et al. 2007).
Many of the licensed salmon sites were not populated even before the outbreak, with
only 12 stocked sites at the time of confirmation. Five uninfected sites in MA 3a were
depopulated as a result of normal harvesting operations within 4 weeks of the
outbreak being detected. This fortunate coincidence greatly reduced the potential
susceptible population within the MA. Following depopulation of the confirmed sites,
which are fallowed for 6 months and suspect sites which are fallowed for 3 months,
the control area will be subject to a co-ordinated fallowing period of 6 weeks before
the processes of assuring the restoration of ISAV freedom can begin. Confirmation
of ISAV freedom will involve a 2 year period of surveillance and diagnostic testing of
all the stocked sites in the surveillance zone, after which MA 3a can be adsorbed
back into the ISAV free zone that incorporates the rest of Great Britain.
3.6.2. Subclinical Infection
The case of site F (and possibly D) suggests subclinical infection may persist
undetected on sites for months. Alternatively there could be an environmental
reservoir, if so longer fallowing may be required for eradication, or an independent
evolution of virulence may have occurred (viral sequencing is planned, to answer this
question); it is unlikely that there would be two random emergence events in MA 3a
within a year, and none elsewhere in Scotland in a decade.
The MA wide movement restrictions and fallowing of sites, following clearance of
confirmed infected sites, help manage the risk from undetected subclinical
populations (provided there is at least one known ISA case in the area). Even if such
unknown infection exists fish will not be moved off and shedding will limited in the
absence of clinical disease (Gregory et al. 2009). However, infection may still spread
e.g. with escapes.
Synchronised fallowing of only control zones rather than whole MAs may represent a
serious risk to attempts to stamp out infection. Subclinical infection could persist in
other parts of the MA and flare up, and involve repopulated sites. There is a good
case for area wide fallowing, post harvest, but this is not currently a requirement
before restocking. In the case of this outbreak prolonged synchronous fallowing was
planned and did occur throughout the winter.
Diagnostic sampling in the absence of clinical signs may be required to detect ISAV
infection. However, if transmission risk is minimised by MA-wide controls then
ignorance of the location of infection may not be too serious. It might be that
38
diagnostic screening of all populated sites in the MA may reduce the risk from not
carrying out synchronous fallowing.
The most serious implication of prolonged periods of asymptomatic infection occurs
when no known cases of infection exist, and hence the area is not subject to
movement restrictions or to enhanced surveillance. It is for this reason that
movements of fish between different MAs should be minimised in the absence of
known cases of ISA. Following depopulation of the confirmed sites, which are
fallowed for 6 months and suspect sites which are fallowed for 3 months, the control
area is subject to a co-ordinated fallowing period of 6 weeks before the processes of
assuring the restoration of ISA freedom can begin. In this case a longer fallowing
was achieved because following the clearance of the last site on 25 November 2009
restocking did not commence until spring 2010, so a 3-4 month fallowing occurred.
3.6.3. Practices Contributing to the Potential Future Re-Emergence of ISA
Virulent ISAV might re-emerge from local avirulent HPR0 or be imported (Murray and
Peeler 2005). The HPR0 strain was found on 3 of 30 anonymously surveyed
Scottish salmon farms (McBeath et al. 2009); however its presence is not associated
with clinical ISA disease. Furthermore ISAV has been detected from wild salmonid
fish in Scotland (Raynard et al. 2001; Cunningham et al. 2002). It is therefore
unlikely that the eradication of HPR0 from Scottish salmon farms can be cost
effective although co-ordinated fallowing will help keep prevalence low. However, as
long as HPR0 is present there remains the potential for ISA disease to re-emerge
(Cunningham et al. 2002). Utilising local well boats for local farm activities such as
harvesting is a good practice and should continue. However, it is possible that ISAV
could be imported with other vessels or even escaped or wild fish. It is also possible
there is a risk of ISA emergence due to use of imported ova from Norway, however
opinion on vertical transmission is divided. Since eradication of HPR0 is not
practicable and contact risk for import of foreign virus is low, there is little that can be
done to further reduce exposure risk, unless the risk of vertical transmission is
significant. Since there is no evidence that the 1998/9 or the 2008/9 ISA outbreaks
were linked to vertical transmission, this means that in the future there is a high
likelihood that ISA will recur even if the import of ova ceased (although the time taken
for this to occur might be increased).
In the event that ISAV is introduced, the emergence of disease is encouraged if the
virus can circulate in populations of fish at high levels for long time periods. This
circulation is broken by fallowing. Fallowing is currently carried out at the farm level
but for it to be more effective in breaking local infection cycles, MAs should be
synchronously fallowed. The operators of the fish farms within MA 3a were
progressing towards a synchronous fallowing strategy however this had not yet been
implemented. Due to the fact that ISAV seems to have persisted undetected on site
F for many months, in spite of frequent visits by FHI, anything other than
39
synchronous fallowing could allow such hidden infection to transmit to newly input
smolts on neighbouring sites. The emergence of multiple disease problems at the
same time (sea lice, SAV and BKD) in addition to ISA further illustrates the need for
synchronisation. The 1998/9 outbreak began in a site containing multiple
generations of fish (Stagg 2003) underlining the need for fallowing.
If ISA were to re-emerge, it is imperative that there is a quick response by industry
and the regulatory authorities; this requires rapidly detecting the presence of ISAV.
Neither the 1998/9 nor the 2008/9 index cases were detected by active surveillance.
There simply can never be enough FHIs to visit sites frequently enough to detect
ISAV within a short time from an outbreak beginning, in the absence of information to
target particular sites. However, passive surveillance has also been disappointing
with ISA only reported in 1998 once high levels of mortality had begun and the virus
was widely dispersed (Murray et al. 2002) and in 2008 mortality was ascribed to high
sea lice burdens. In 2008 inspectors did target the putative index site following up
reports of mortality and took samples for diagnostic screening, even although there
was an alternative explanation for the mortality. This use of intelligence-led sampling
indicates that reporting of unusual mortality even if it is ‘explained’ could be a
valuable tool to more effectively targeting Marine Scotland’s resources. There is
provision for an element of intelligence led surveillance in Scotland’s current risk
based surveillance programme.
Once ISAV was detected on the putative index site active surveillance of the
remaining sites in MA 3a, and sites in other MAs identified as having potential
contact with MA 3a, was effective in detecting further ISA cases. This is because the
risk-based targeting of surveillance effort allowed these sites to be frequently visited.
Site E was detected by passive surveillance, with suspicion of ISA being reported by
a veterinarian.
A highly important factor in the limitation of ISA in 2008/9 was the MA structure
developed following the 1998/9 outbreak (Stagg 2003). MAs must be
epidemiologically appropriate, and the spread of ISAV between smaller MAs that
were defined in the Code of Good Practice (CoGPWG 2006) showed these to be
ineffective. A MA can never be completely biosecure and so infection may spread
while infected fish remain, even after movements of live fish have been prevented.
Movements of live fish between MAs (Figure 6) should not be undertaken, even in
the apparent absence of infection. This is because infection can be spread if there is
any delay between emergence and detection as occurred in 1998 (Stagg 2003).
Another important factor was the disinfection of processing plant effluent and the
disinfection and sealing of well boats (CoGPWG 2006). All waste from processing
plants was disposed of by approved methods. From investigation, there is little
evidence to associate well boats with the spread of ISAV, unlike in 1998/9 (Murray et
al. 2002) and this has been a major achievement in reducing Scotland’s vulnerability
40
to ISA outbreaks. However preventing the risky practice of bus-stop deliveries could
further reduce the risks.
Synchronous fallowing of MAs is likely to substantially reduce the risk of the
emergence of ISA, however the risk cannot be eliminated. Epidemiologically
appropriate MAs have been successful in confining the virus. It is likely impossible to
prevent hydrodynamic spread within MAs, while open culture net-pens are used to
farm salmon. Movement of fish between sites in different MAs and bus-stop
deliveries are identified as risky practices that should be prevented or if this is
unfeasible then carefully controlled. Disinfection of processing plant effluent and the
use of local dedicated well boats are good practices that should be encouraged.
4. Conclusions
An outbreak of ISA has occurred in Scotland. The outbreak is of unknown origin, but
was not related to the ISAV that was responsible for the 1998/9 outbreak. Whether
ISA evolved from a local avirulent HPR0 virus or was imported via an unknown route,
such a re-emergence may be repeated and so, even on eradication, complacency
cannot be allowed in aquaculture biosecurity. Policies such as co-ordinated fallowing
of areas and risk based surveillance may deny emergent or introduced virus a
chance to establish.
A rapid response to the outbreak by Marine Scotland was possible, because of rapid
detection of infection. Detection at site A occurred as a result of intelligence from an
unconfirmed source in relation to alleged high sea lice mortality in Shetland and
inspectors following up general health problems, not specifically targeting ISA. This
observation justifies the need for Marine Scotland to investigate reports of mortality,
even if ascribed to a non-notifiable cause, and indicates the limits of passive
surveillance for notifiable diseases in the face of multiple potential causes of
mortality. Intelligence led surveillance plays an important role in Marine Scotland’s
current risk based surveillance programme. Active surveillance is of limited
effectiveness unless the limited available effort can be targeted in a risk-based
manner. Even then, subclinical infection may go undetected and so there may be a
case for regular diagnostic sampling (instead of just the current one off sample) in the
absence of signs of ISA if sites are likely to have been exposed to infection.
Fallowing is important in eliminating ISAV and other diseases and in preventing their
emergence in the first place. Currently there is no requirement to fallow whole MAs
synchronously, only control zones and this practice may allow subclinical infection to
persist. Surveillance zones within a protected area do fallow for 6 weeks
asynchronously, which will have a positive effect on minimising re-emergence of
disease. Fortunately, all sites in MA 3a were fallowed for the whole winter of
2009/10.
41
The outbreak was confined to a relatively small area, although the virus spread easily
within that area. The strategic breaking of the industry into biosecurity-determined
management areas by Marine Scotland Science for the purpose of disease control
appears to have been effective at controlling ISAV. Isolation has been maintained
because of an absence of seawater-to-seawater movements of fish in the critical
period and the disinfection of processing plant waste and well boats. Depopulation of
confirmed sites prevents these from acting as sources of infection.
The industry has played a key role in the design of these effective disease control
measures (Anon 2000; CoGPWG 2006); this has been done in collaboration with the
Scottish Government; at significant financial cost to industry and the government.
There have been serious failings in these controls, seawater-to-seawater movements
were quite extensive in the first six months of 2008, and if an outbreak had occurred
at that time, ISAV could have spread much more widely, at least throughout
Shetland. Similarly, harvest vessels practised ‘bus-stop’ harvests, also potentially
risking spread at the regional scale. Some depopulation has been delayed owing to
technical or other reasons, allowing large numbers of diseased fish to remain in the
environment for considerably longer than the mean 3-4 week period. These detected
failings suggest industry practice could be improved to further reduce the risk from
any future ISA outbreaks, especially before infection was detected, and also reduce
the probability of outbreaks occurring in the first place. These failings aside, industry
behaviour combined with the speedy response of Marine Scotland, has played a key
role in the confinement of ISAV relative to spread in other countries, or indeed
Scotland in 1998/9.
The disease control mechanisms that appear effective at controlling ISAV in Scotland
help to control other diseases. However, the contact structure involving freshwater
sites spreads throughout the UK, compared to the networks of marine site-to-site
movement (local and limited) and of harvest (regional). As such, diseases with
freshwater spread are likely to be more difficult to control. Similarly more robust
pathogens might transmit over greater distances and so be more likely to transmit
between MAs. With suitable modifications, the system of local MAs with associated
good biosecurity measures that appears to have been successful in the control of
ISA, and refined in the light of epidemiological information, may be applied to the
control of a range of diseases.
5. Acknowledgements
The data used for this report was derived from samples collected by Fish Health
Inspectors and scientists working for, and funded by, Marine Scotland Science and
was analysed by diagnostic scientists working in the Marine Scotland Science Marine
Laboratory in Aberdeen. We thank Rachel Kilburn, Campbell Pert, Sonia McBeath,
42
Katy Urquhart, Paul Cook, Anna Turnbull, Nicola McManus, Una McCarthy, Warren
Murray, Mickael Fourrier, Kelly MacNeish, Sarah Weir, Darryl McLennan, Nicola
Bain, Julia Black, Fiona Doig and the Fish Health Inspectors – Amanda Walker,
Andrea Warwick, Andrew Mayes, Daniel Stewart, David Tomlinson, Jacqueline
Parker, Kate Smith, Katy A Urquhart, Mark Paterson, Neil Purvis, Paul McKay, Ron
Smith, Sandra Johnson, Sebastien Rider and Sonia Duguid – for their contributions.
Data on movement of ships, operation of harvest plants and mortality data from farm
sites were provided by the respective industries. Industry cooperation contributed
significantly to the control of ISA in the Shetland Islands.
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190.
48
Appendix 1 Details of Diagnostic Methods: Eann Munro
As detailed in the Office international des épizooties (OIE), Manual of Diagnostic
Tests for Aquatic Animals 2009, the diagnosis of ISA is based on clinical and
pathological findings as well as detection of the agent. The diagnostic methods are
summarised in this appendix 1’s figure 1.
Figure 1 Diagnostic test procedures for confirmation of ISA
Gross pathological and histopathological are changes that are detailed in the Manual
of Diagnostic Tests for Aquatic Animals (OIE, 2009).
49
Agent Detection and Identification Methods
Indirect Fluorescent Antibody Test
An indirect fluorescent antibody test (IFAT) using a validated monoclonal antibody
(MAb, 3H6F8; NVI, Norway) against ISAV haemagglutinin-esterase (HE) on kidney
imprints is performed on a case where clinical signs consistent with ISA disease are
observed by Marine Scotland fish health inspectors.
Preparations of Kidney Imprints
In the field, a section of mid-kidney is briefly blotted against absorbent paper to
remove excess fluid and blood, and imprints blotted on poly-L-lysine-coated
microscope slides. The imprints are air-dried, fixed in acetone/methanol (1:1 v/v) for
5 min and stored at 4°C for a maximum of 72h until use.
Staining Procedure
After blocking with 6% skimmed milk in phosphate-buffered saline (PBS) containing
0.2% tween 20 (PBSt) for 30 min, the samples are incubated for 1 h with a 1:100
dilution of anti-ISAV MAb (3H6F8) in 1% skimmed milk, followed by three washes.
For the detection of bound antibodies, the imprinted slides are incubated with
fluoescein isothiocyanate (FITC)-conjugated anti-mouse Ig (Sigma-Aldrich, UK) at a
1:1000 dilution in 1% skimmed milk for 1 h. The slides are again washed three times
before incubation with CITIFLUOR AF1 solution (CITIFLUOR Ltd., UK) at a (1:4 v/v
dilution in PBS). A further three washes are performed and the slides counter
stained with propidium iodide (0.01mg/ml) for 3 min. The slides are once again
washed 3 times and a coverslip placed on the slides aided by VECTASHIELD®
Hard-Set™ (Vector Laboratories Inc., USA) mounting medium. PBS with 0.1%
Tween 20 is used for all washing steps, all incubations are performed at room
temperature and the microscope slides are kept in the dark until reading commences.
Slides are read using an Olympus BX60 upright fluorescent microscope (Olympus
America Inc, USA).
Immunohistochemistry
Tissue material for histological examination is sampled in the field and returned to the
Marine Laboratory for processing. Paraffin sections from formalin-fixed tissues are
prepared according to standard histological methods. Slides containing gill; gut;
heart; kidney; liver; spleen, pancreas and skin tissue, along with confirmed positive
and negative control slides are prepared for immunohistochemistry (IHC), where a
monoclonal antibody against the ISAV nucleoprotein (MAb P10; Aquatic diagnostics
Ltd., UK) is used to detect the presence of the virus within the tissue. Stained slides
50
are subsequently analysed by microscopy and the presence of ISAV is indicated by
red staining of the tissue.
Preparation of Tissue
The tissues (gill; gut; heart; kidney; liver; spleen, pancreas and skeletal muscle) are
fixed in neutral phosphate-buffered 10% formalin for a minimum of 24h, dehydrated
in graded ethanol, cleared in xylene and embedded in paraffin, according to standard
histological protocols. Approximately 3 µm thick sections are cut onto poly-L-lysine-
coated slides and heated at 45–50°C for a minimum of 12 h, de-waxed in clearene
and rehydrated through graded ethanol.
Staining Procedure for IHC
All incubations are carried out at room temperature on a rocking platform, unless
otherwise stated. A glass pen is used to circumscribe the sections on the glass
slides, to allow better adherence of the ImmEdge pen (Vector Laboratories Inc.,
USA) once the slides are wet. Endogenous peroxidase activity within the tissues is
blocked using 3% hydrogen peroxide solution in TBS for 5 min. Slides are washed
once in Tris buffer saline (TBS) and once in TBS with 0.1% Tween 20 (TBSt). The
slides are then blocked using 1% horse serum from VECTASTAIN® Elite ABC kit
(Universal) (Vector Laboratories Inc., USA) to prevent non specific binding of the
MAb. Excess liquid is removed and the slides are incubated with the anti-ISAV MAb
(P10) at a 20µgml-1 solution in TBS for 1h. The slides are washed three times in
TBSt and incubated with a secondary biotinylated horse antibody solution from the
ABC kit for 30 min as per manufacturer instruction. The slides are once again
washed in TBSt and covered with a peroxidise Avidin-Biotin Complex (ABC) reagent
for 30 min. After this step, the slides are washed twice in TBSt and once with TBS
and incubated with AEC staining solution (Sigma-Aldrich, UK) for 10min to detect the
presence of peroxidise within the ABC/antibody bound complex. The slides are
placed in distilled water for 5min and counterstained with Haematoxylin QS (Vector
Laboratories Inc., USA) for 2min with excess stain removed under running tap water.
Coverslips are placed onto the slides using CITIFLUOR mounting medium
(CITIFLUOR Ltd., UK). Before reading, the slides are left for a minimum of 1 h to
allow the blueing process to occur. All slides are read using an Olympus BX60
upright microscope under brightfield conditions.
51
Agent Isolation and Identification
Cell Culture Isolation
Inoculation of cell monolayers
Approximately 1g of mixed tissue sample (kidney, liver, spleen, heart) is aseptically
dissected into 9 mL of viral transport media (Liebovitz L-15 cell culture media
supplemented with 10% newborn calf serum, gentamicin (50mg/ml), polymixin ‘B’
(10,000U/ml); pH 7.4 – 7.8) in the field by Marine Scotland fish health inspectors and
transported chilled to the Marine Laboratory. The VTM solution containing the tissue
samples are homogenised within 48 h of sampling using an Enfer ETDS VIII tissue
homogeniser (Design Village, UK), clarified by centrifugation and the supernatant
sterile filtered before being mixed at a 1:1 v/v ratio with infectious pancreatic necrosis
virus neutralising polyclonal antiserum and incubated at 15oC for 1-2 h. The
sample/IPNV antisera solutions are inoculated at a 1:1000 dilution (final dilution of
tissue material to culture medium) onto 12 well plates containing a monolayer of TO
cells (Wergeland H., Department of Fisheries and Marine Biology, University of
Bergen). The TO cell monolayers are at a confluency of 60 – 80%, 24 – 48 h old and
incubated at 15oC after inoculation. A negative cell control and a 6 serial dilution
positive control (10-3 – 10-8 dilution series), using the Scottish ISAV reference isolate
(390/98) are prepared on the days of sample inoculation and subcultivation.
Monitoring Incubation
Growth of the virus may lead to a cytopathic effect (CPE) which is observed
microscopically. Inoculated TO cultures are examined every 7 days for the
appearance of CPE. If CPE occurs, an aliquot of the cell culture medium is screened
for ISAV by IFAT. If IPNV is identified as the causative agent of the CPE, fresh TO
cultures are inoculated with tissue homogenate supernatant (previously stored at -80
oC) incubated with a lower dilution of IPNV antisera. If no CPE has developed after
14 days, the cell monolayers are screened by haemadsorption and subcultivated to
fresh TO cultures.
Subcultivation Procedure
Aliquots of culture media from the primary culture plates are inoculated onto 12 well
plates containing fresh TO cells (culture conditions as above) at a 1:10 dilution 14
days (or earlier when obvious CPE appears) after inoculation. The secondary
cultures are incubated at 15 oC for 14 days and CPE monitored as detailed for
primary cultures. At the end of the incubation period, or earlier if obvious CPE
appears, the medium is collected for virus identification (IFAT). All primary and
secondary cultures, including those that demonstrate no CPE and all positive and
52
negative control wells, are examined for the presence of ISAV by haemadsorption
after the 14 day incubation period (or earlier when obvious CPE appears).
Haemadsorption Assay
In the absence of CPE, a haemadsorption test is performed to detect intra-cellular
virus. Atlantic salmon erythrocytes are overlayed on the drained cell monolayer. If
the TO cells are virus-infected the erythrocytes will adhere to the cell monolayer in
discrete foci or haemadsorption centres. This test gives a presumptive identification
of a fish myxovirus. Briefly, blood from Atlantic salmon is freshly collected from the
Marine Laboratory stock aquaria and centrifuged to pellet the erythrocytes. The red
blood cells are then diluted in cell culture media at a 1:2000 dilution. The cell culture
media is removed from each of the sample wells, 0.5 mL of the erythrocyte/media
suspension is gently layer onto the TO cell monolayer and the cultures are incubated
at room temperature for 45 min. After this incubation period, the erythrocyte/media
suspension is discarded and the cell monolayers washed three times in cell culture
media prior to the cultures being read under an inverted microscope. ISA virus from
a positive haemadsorption sample is identified by an immunofluorescent antibody
test (IFAT).
Immunofluorescent Antibody Test
TO cell monolayers are freshly prepared in 24 well plates at a confluency of 60 –
80%, 24 – 48h old. TO cell monolayers are inoculated in duplicate with sample
media supernatant at 1:5 and 1:25 dilutions and incubated at 15oC for 4-7 days.
Duplicate negative cell controls are included on each sample and positive control
plates. Positive control plates are set up as described above but using the neat ISAV
Scottish reference strain 390/98.
Once CPE occurs on the TO monolayer, the cell culture media is removed and the
cell monolayer rinsed once with PBS and fixed in 80% acetone for 20 min. The
acetone is then discarded and the plates left to air dry for 1 h. The fixed cell
monolayers are then incubated at 37oC for 30 min with anti- ISAV MAb (MAb mix;
MAb 3H6F8 + 10C9F5, NVI, Norway) at either a 1:20 or 1:50 dilution in PBS. The
anti- ISAV MAb is then removed and the culture is rinsed three times with
PBS/0.05% Tween 20 (PBSt). A secondary FITC-conjugated goat anti-mouse (Ig)
antibody (Sigma-Aldrich, UK), diluted 1:100 in PBS is applied to the fixed monolayer
and incubated at 37oC for 30 min. The secondary antibody is removed and the
monolayer rinsed a further three times with PBSt. TO cultures are examined using a
Nikon diaphot UV inverted microscope (Nikon Corp, Japan).
53
Real-Time Reverse-Transcription Polymerase Chain Reaction
RNA Extraction
Kidney material (approximately 0.25g) for molecular examination is sampled into 1
mL of RNALater® (Sigma-Aldrich, UK) in the field and returned chilled to the Marine
Laboratory for processing. Samples are homogenised using a tissue lyser (Qiagen,
UK). Total RNA from kidney tissue (5 mg) is extracted using the fully automated
BioRobot M48 (Qiagen, UK) which elutes 100µL of total RNA in a pre-defined volume
of elution buffer. Negative extraction controls were conducted by performing a blank
extraction. Controls were taken through subsequent RT and PCR steps.
Reverse Transcription
cDNA from the extracted RNA and the negative extraction control was generated
using ABI Taqman® Reverse Transcription Reagents kit with random hexamers
(Applied Biosystems, UK). Approximately 1.0µg total RNA in a volume of 7.7µl is
added to 12.3 µl of the reverse transcriptase mixture, giving a final volume of 20µl
containing 1x RT buffer (25mM Tris-HCl, pH 8.3, 37.5mM KCl), 5.5mM MgCl2, 0.5mM
of each deoxynucleoside triphosphate (dNTP), 1.25uM random hexamers, 0.4U
RNase Inhibitor and 1.25 U multiscribe reverse transcriptase. The mixture is
incubated for 10 min at 25°C, 30 min at 48oC, heat inactivated at 95oC for 5 min. A
negative RT control reaction is performed replacing the RNA with RNAse free-water
and taken through subsequent PCR step. A positive control is also included in the
assay, which can be distinguished from sample material in the Real-time PCR.
Real-Time PCR
RT-PCR primer pairs and Taqman® MGB probes (Applied Biosystems, UK)
targeting ISAV segment 8 and the Atlantic salmon elongation factor 1 alpha
reference gene (ELF1-α) are used for the diagnostic real-time assay. Both primers
and probes for ISAV are targeted to conserved gene regions to ensure detection of
all documented variants of ISAV. All probes were designed over an intron-exon
splicing site to exclude amplification from potential genomic contamination. In a
diagnostic context, the ELF 1- α controls, provide a powerful quality control of
individual samples. Assaying for constitutively expressed endogenous genes also
allows for quantification of pathogen nucleic acid levels and facilitates direct
comparison of relative levels of pathogen detected between samples.
54
Quantitative PCR is performed on an ABI Prism 7000 Sequence Detection System
(Applied Biosystems). The assays are carried out in a 20µl volume, containing
900nM of each primer, 250nM Taqman® probe, 1 x PCR SensiMix (dU), 2.5 mM
MgCl2, 1 x Uracil DNA Glycosylase (UNG) (Quantace), dH2O and 1µl cDNA. The
following universal cycling conditions are applied to ISAV diagnostic samples:-
Step Temp Time Number of Cycles
1 37oC 10 Minutes 1 Cycle
2 95oC 10 Minutes 1 Cycle
3 95oC
60oC
15 Seconds
1 Minute
45Cycles
No-target controls (NTCs) were performed for each primer and probe set. The
primers and probes utilised in this assay are detailed below.
Assay
Name Forward Primer Reverse Primer Probe Positive
Control Probe
ISAVS
EG8
ISAVSEG8UNIF
5’- CTACACAGC
AGGATGCAG
ATGT-3
ISAVSEG8UNIR
5’-CAGGATGCCGG
AAGTCGAT-3’
ISAVSEG8UNIM
2
6-FAM-
CATCGTCGCT
GCAGTTC-MGB
ARTIFICIAL
PROBE
6-VIC-
ACCGTCTAGC
ATCCAGT-
MGB
ELF
RNA
Salmon
EL.1A-
ELAFSALM-F
5’-
CCCCTCCAGGA
CGTTTACAAA-3’
EL.1A-ELARSALM-
R
5’-
CACACGGCCCAC
AGGTACA-3’
EL.1A-ELAM1
6-FAM-
ATCGGTGGTA
TTGGAAC-MGB
N/A
55
Appendix 2 Complete Testing Histories of Confirmed Sites
Two tables are presented that include all diagnostic test results obtained from all
samples taken by FHI from within MA 3a (Table 1) and from outside MA 3a but within
Shetland (Table 2).
Appendix 2 Table 1
Complete testing history for sites in MA 3a from 9/12/08 to fallowing on 26/11/2009
Site No Site Name Inspector Case No Case Type Date Of Visit Frequency Result type
FS0673 East of Hildasay Danny Pendrey 20080974 Diagnostic 09/12/2008 1/1 ISAP
FS0673 East of Hildasay Danny Pendrey 20080982 Statutory sample (eg 150-fish test) 16/12/2008 8/30 KPCR
FS0673 East of Hildasay Danny Pendrey 20080974 Diagnostic 09/12/2008 1/1 ISAH
FS0673 East of Hildasay Danny Pendrey 20080982 Statutory sample (eg 150-fish test) 16/12/2008 7/30 ISAV
FS0936 Foreholm P aul McKay 20 090063 Statutory sample (eg 150-fish test) 06/01/2009 0/30 KPCR
FS0936 Foreholm P aul McKay 20 090063 Diagnostic 06/01/2009 0/30 ISAV
FS0515 North Papa Paul McKay 20090064 Diagnostic 07/01/2009 0/1 KPCR
FS0447 Papa Paul McKay 20090065 Diagnostic 07/01/2009 0/2 KPCR
FS0935 Flotta Yvonne McMurchie 20090043 Diagnostic 07/01/2009 0/2 KPCR
FS0316 Setter V oe Ron Smith 20090124 D iagnos tic 07/01/2009 1/2 KPCR
FS0316 Setter V oe Ron Smith 20090124 D iagnos tic 07/01/2009 0/2 ISAV
FS0691 Sound of Hoy Yvonne McMurchie 20090044 D iagnostic 08/01/2009 0/2 KPCR
FS0937 W est of Burwick Paul McKay 20090067 Diagnostic 08/01/2009 0/1 KPCR
FS0785 Spoose H olm Ron Smith 20090138 Diagnostic 09/01/2009 0/1 KPCR
FS0447 Papa Katy Urquhart 20090364 Statutory sample (eg 150-fish test) 12/01/2009 4/30 KPCR
FS0433 Langa Isle (East) Danny Pendrey 20090365 Statutor y sample (eg 150-fish test) 12/01/2009 0/30 KPCR
FS0447 Papa Katy Urquhart 20090364 Statutory sample (eg 150-fish test) 12/01/2009 8/30 ISAV
FS0316 Setter Voe K aty Urquhart 20090376 Statutory sample (eg 150-fish test) 13/01/2009 0/30 ISAP
FS0937 W est of Burwick Paul McKay 20090367 Statutory sample (eg 150-fish test) 13/01/2009 0/30 KPCR
FS0316 Setter Voe K aty Urquhart 20090376 Statutory sample (eg 150-fish test) 13/01/2009 0/30 ISAV
FS0674 North Havra Paul McKay 20090368 Statutory sample (eg 150-fish test) 14/01/2009 0/30 KPCR
FS0515 North Papa Katy Urquhart 20090369 S tatutory sample (eg 150-fish test) 14/01/2009 0/30 KPCR
FS0785 Spoose Holm Katy Urquhart 20090366 Statutor y sample (eg 150-fish test) 15/01/2009 0/5 KPCR
FS0785 Spoose Holm Sonia Duguid 20090244 Statutory sample (eg 150-fish test) 16/01/2009 0/30 KPCR
FS0935 Flotta Sonia Duguid 20090371 Statutory sample (eg 150-fish test) 17/01/2009 0/30 KPCR
FS0320 Loch of Strom Danny Pendrey 20 090164 Statutory sample (eg 150- fish tes t) 29/01/2009 0/30 KPCR
FS0548 Lea Trondra (East of Trondra) Yvonne McMurchie 20090048 S tatutory sample (eg 150-fish test) 04/02/2009 0/30 ISAP
FS0548 Lea Trondra (East of Trondra) Yvonne McMurchie 20090048 S tatutory sample (eg 150-fish test) 04/02/2009 0/30 ISAV
FS0433 Langa Isle (East) Paul McKay 20 090075 Diagnostic 10/02/2009 0/1 KPCR
FS0433 Langa Isle (East) Paul McKay 20 090075 Diagnostic 10/02/2009 0/1 ISAV
FS0447 Papa Paul McKay 20 090081 Diagnostic 11/02/2009 1/3 KPCR
FS0447 Papa Paul McKay 20 090081 Diagnostic 11/02/2009 2/2 ISAI
FS0447 Papa Paul McKay 20 090081 Diagnostic 11/02/2009 1/3 ISAV
FS0447 Papa Danny Pendrey 20090178 Researc h (eg FCB aquarium) 05/03/2009 0/10 KPCR
FS0447 Papa Danny Pendrey 20090178 Researc h (eg FCB aquarium) 05/03/2009 0/10 GPCR
FS0447 Papa Danny Pendrey 20090178 Researc h (eg FCB aquarium) 05/03/2009 0/60 IS AV
FS0515 North Papa Danny Pendrey 20090180 D iagnos tic 11/03/2009 2/2 ISAP
FS0515 North Papa Danny Pendrey 20090180 D iagnos tic 11/03/2009 2/3 ISAH
FS0515 North Papa Danny Pendrey 20090180 D iagnos tic 11/03/2009 2/2 ISAV
FS0515 North Papa Danny Pendrey 20090184 D iagnos tic 18/03/2009 1/2 ISAP
FS0515 North Papa Danny Pendrey 20090184 Stat utory sample (eg 150-fish test) 18/03/2009 16/30 ISAP
FS0515 North Papa Danny Pendrey 20090184 D iagnos tic 18/03/2009 12/12 ISAI
FS0515 North Papa Danny Pendrey 20090184 Stat utory sample (eg 150-fish test) 18/03/2009 15/30 ISAV
FS0433 Langa Isle (East) Paul McKay 20 090508 Diagnostic 14/05/2009 2/2 ISAI
FS0433 Langa Isle (East) Paul McKay 20 090508 Diagnostic 14/05/2009 1/1 KPCR
FS0433 Langa Isle (East) Paul McKay 20 090508 Diagnostic 14/05/2009 5/5 ISAA
FS0433 Langa Isle (East) Paul McKay 20 090508 Diagnostic 14/05/2009 1/1 ISAV
FS0548 Lea Trondra (East of Trondra) Danny Pendrey 20090198 Diagnostic 19/05/2009 3/3 ISAP
FS0548 Lea Trondra (East of Trondra) Danny Pendrey 20090198 Statutor y sample (eg 150-fish test) 19/05/2009 25/30 ISAP
FS0548 Lea Trondra (East of Trondra) Danny Pendrey 20090198 Diagnostic 19/05/2009 6/12 ISAI
FS0548 Lea Trondra (East of Trondra) Danny Pendrey 20090198 Diagnostic 19/05/2009 12/12 ISAA
FS0548 Lea Trondra (East of Trondra) Danny Pendrey 20090198 Diagnostic 19/05/2009 3/3 ISAV
FS0548 Lea Trondra (East of Trondra) Danny Pendrey 20090198 Statutor y sample (eg 150-fish test) 19/05/2009 27/30 ISAV
FS0316 Setter V oe Paul McKay 20090961 Diagnostic 28/10/2009 1/1 ISAV
FS0316 Setter V oe Paul McKay 20090961 Diagnostic 28/10/2009 1/1 ISAA
FS0316 Setter V oe Paul McKay 20090961 Diagnostic 28/10/2009 1/1 KPCR
FS0316 Setter V oe Sonia Duguid 20091021 Research (eg FCB aquarium ) 09/11/2009 0/2 ISAV
Frequency (2nd last column is the number of positive pools divided by the total
number of pools in the sample. Result type (last column of each table) codes are:
ISAA = ISA (histology); ISAH ISA Immunohistochemistry (IHC); ISAG = ISA PCR (gill
sample); ISAI = ISA (IFAT); ISAP = ISA PCR (kidney); ISAV = ISA virus isolation;
56
KPCR = ISA real time qPCR (kidney); GPCR = ISA real time qPCR (gill). These
diagnostic tests are described in appendix 1.
Appendix 2 Table 2
Complete testing history for sites in Shetland outside MA3a during 2009. Note that
all results are negative.
Site No Site Name Inspector Case No Case Type Date Of V isit Frequency Res ult type
FS0666 B urrastow Paul McKay 20090079 D iagnostic 28/01/2009 0/1 KPCR
FS0620 A ith (East) Yvonne McM urchie 20090050 Statutory sampl e (eg 150-fish test) 02/02/ 2009 0/30 KPCR
FS0620 A ith (East) Yvonne McM urchie 20090050 Statutory sampl e (eg 150-fish test) 02/02/ 2009 0/30 ISAV
FS1053 Hogan Andrea Warwick 20090324 Statutory sampl e (eg 150-fish test) 03/02/ 2009 0/30 KPCR
FS1053 Hogan Andrea Warwick 20090324 Statutory sampl e (eg 150-fish test) 03/02/ 2009 0/30 ISAV
FS0666 B urrastow Andrea Warwick 20090325 Statutory sampl e (eg 150-fish test) 05/02/ 2009 0/30 KPCR
FS0397 Dale s Voe Y vonne McMurchie 20090049 Statutor y sample (eg 150- fish test) 05/02/ 2009 0/30 KPCR
FS0397 Dale s Voe Y vonne McMurchie 20090049 Statutor y sample (eg 150- fish test) 05/02/ 2009 0/30 ISAV
FS0666 B urrastow Andrea Warwick 20090325 Statutory sampl e (eg 150-fish test) 05/02/ 2009 0/30 ISAV
FS0397 Dale s Voe D anny Pendr ey 20090421 Diagnostic 06/04/2009 0/1 ISAP
FS0397 Dale s Voe D anny Pendr ey 20090421 Diagnostic 06/04/2009 0/1 ISAV
FS0501 Tai ng of Railsbrough C atfirth D anny Pendr ey 20090431 Diagnostic 08/04/2009 0/1 ISAV
FS0501 Tai ng of Railsbrough C atfirth D anny Pendr ey 20090431 Diagnostic 08/04/2009 0/1 ISAP
FS0608 V idli n North Danny Pendrey 20090191 Diagnostic 29/04/ 2009 0/1 ISAP
FS1043 Nes s of Copister Paul McKay 20090509 D iagnostic 25/05/2009 0/1 KP CR
FS1043 Nes s of Copister Paul McKay 20090509 D iagnostic 25/05/2009 0/1 IS AV
FS0088 Clou din Andrea Warwi ck 20090633 Diagnostic 15/06/2009 0/2 KPCR
FS0088 Clou din Andrea Warwi ck 20090633 Diagnostic 15/06/2009 0/2 ISAV
FS0814 S warta Skerry Sonia Duguid 20090569 Diagnostic 07/07/2009 0/1 KPCR
FS0814 S warta Skerry Sonia Duguid 20090569 Diagnostic 07/07/2009 0/1 ISAV
FS0079 B rindister Katy Urquhart 20090686 D iagnostic 05/08/2009 0/1 KPCR
FS0715 Li nga Katy Urquhart 20090685 Diagnostic 05/08/2009 0/2 GPCR
FS0715 Li nga Katy Urquhart 20090685 Diagnostic 05/08/2009 0/2 ISAV
FS0079 B rindister Katy Urquhart 20090686 D iagnostic 05/08/2009 0/1 ISAV
FS1053 Hogan D anny Pendrey 20090782 D iagnostic 18/08/2009 0/1 KPCR
FS0166 Holm of Gruting Danny Pendrey 20090783 Diagnos tic 18/08/2009 0/1 KP CR
FS0167 M id Taing D anny Pendrey 20090784 D iagnostic 18/08/2009 0/1 KP CR
FS0167 M id Taing D anny Pendrey 20090784 D iagnostic 18/08/2009 0/1 IS AV
FS0166 Holm of Gruting Danny Pendrey 20090783 Diagnos tic 18/08/2009 0/1 IS AV
FS1053 Hogan D anny Pendrey 20090782 D iagnostic 18/08/2009 0/1 ISAV
FS0715 Li nga Sonia Duguid 20090577 D iagnostic 24/08/2009 0/1 KPCR
FS0079 B rindister Sonia Duguid 20090578 Diagnostic 24/08/ 2009 0/1 KPCR
FS0079 B rindister Sonia Duguid 20090578 Diagnostic 24/08/ 2009 0/1 ISAV
FS0715 Li nga Sonia Duguid 20090577 D iagnostic 24/08/2009 0/1 ISAV
FS0408 P oseidon D anny Pendrey 20090920 D iagnostic 01/10/2009 0/2 KPCR
FS0408 P oseidon D anny Pendrey 20090920 D iagnostic 01/10/2009 0/2 ISAV
FS1060 W ick of Gart h Mark Paterson 20090878 D iagnos tic 15/10/2009 0/1 KP CR
FS1060 W ick of Gart h Mark Paterson 20090878 D iagnos tic 15/10/2009 0/1 IS AV
FS0397 Dale s Voe Pa ul McKay 20090960 D iagnostic 27/10/2009 0/1 KPCR
FS0183 S outh Sound Paul McKay 20090958 D iagnostic 27/10/2009 0/1 KP CR
FS0183 S outh Sound Paul McKay 20090958 D iagnostic 27/10/2009 0/1 IS AA
FS0397 Dale s Voe Pa ul McKay 20090960 D iagnostic 27/10/2009 0/1 ISAV
57
Appendix 3 Procedure for Notification of Affected Companies
ISA is a notifiable disease in Scotland, as such a representative of fish farm
companies, veterinarians or any other individual who has reason to suspect the
presence of ISA on a site is legally obliged to report this suspicion to Scottish
Ministers. The relevant legislation is now Council Directive 2006/88/EC which
amalgamated and updated the relevant fish health legislation as interpreted under
The Aquatic Animal health (Scotland) Regulations 2009. However, prior to March
2009, The Diseases of Fish Acts 1937 and 1983, EC Directives 91/67/EEC and
amendments e.g. CD 2003/466/EC were in force. Whilst the name of the movement
restrictions imposed under this legislation was changed from DAOs and TDNs to
Initial Designation Notices (IDN) and Confirmed Designation Notices (CDN), the
controls imposed remained similar except that the TDO and DAO did not control the
movement of dead fish or equipment, being reliant of Control Notices for dead fish
and Gate Notices for equipment.
Once informed of a suspicion, or as a result of their own routine surveillance, the FHI
are required to investigate by site inspection investigating by diagnostic sampling
and, where applicable, taking statutory samples. These are taken as soon as
logistically possible following notification; realistically this requires 2-3 working days.
The company representative, as registered with Marine Scotland Science for disease
control purposes, is immediately informed on receipt of any result from the diagnostic
laboratory, by FHI, initially by phone but with a follow up written report.
In the case of a non-definitive result (insufficient to confirm or rule out ISA), as
occurred initially at site A, a Thirty Day Notice (TDN) was served on the site under 4A
of the Diseases of Fish Act 1937, as amended. This allowed official suspicion and
confirmatory testing to be completed.
Where a single diagnostic test, such as PCR is unsupported by further test results
(see Table 1 in main text), the criteria for official suspicion will not be met and
movement restrictions will be lifted from that site on completion of the case. Where a
site is under official suspicion, it remains suspect until six months have passed, in the
absence of further evidence leading to confirmation.
When further laboratory results became available (on 2 January 2009 in the case of
this outbreak) the presence of ISA was confirmed. Companies in the affected area
were informed by phone, fax and email; however as this was a national holiday not
all companies responded to contacts. Backup phone message were left, combined
with emails. All companies were informed as a priority and as soon as practically
possible.
58
A Designated Area Orders (DAO), prepared under the terms of section 2 of the
Diseases of Fish Act 1937, as amended, were prepared for every fish farming site in
MA 3a and faxed to the business correspondent of the companies concerned.
Movement restrictions are placed in the form of: a Confirmed Designation Notice
(CDN), formally Designated Area Order (DAO) which controls the movement of live
fish and food and removal of mortalities; a Gate Notice (GN) which controls
movement of staff, equipment and dead fish, or a Control Notice (CN) which controls
movement of dead fish, are placed on sites within Control and Surveillance Zones.
SGMS issue written notification to each fish farm company within the control and
surveillance zones, detailing movement restrictions on each site.
During the course of the outbreak, subsequent results from tests undertaken on fish
farm sites in MA 3a were communicated to company correspondents by telephone,
and followed up with the issue of a written fish health report, following agreed Fish
Health Inspectorate (FHI) procedures.
Where positive results were obtained from biological or chemical diagnostic testing
the case was reviewed by senior diagnosticians and scientists to consider whether
there was sufficient evidence to confirm or officially suspect the presence of ISA (see
table 1 in main text). The criteria considered are defined by the OIE and also laid
down by the European Commission in Commission Decision 2003/466/EC for the
suspicion of infection with ISA or confirmation of ISA. The purpose of these
reviews was to ensure that the results of the laboratory results were valid and to
measure whether the criteria for suspecting or confirming the presence of ISA had
been met.
Where the presence of ISA was confirmed, this information was passed to Scottish
Government who, on their agreement with the MSS diagnosis, issued a withdrawal
notice and was also communicated to the business correspondent of the relevant
company. Scottish Government then issued a withdrawal notice, initially in
accordance with The Diseases of Fish (Control) Regulations 1994, subsequently
under The Aquatic Animal health (Scotland) Regulations 2009. The MSS decision
and the withdrawal notice were communicated to the business correspondent of the
affected company.
During the outbreak, concern was raised by industry representatives that insufficient
information was being passed to affected companies with regard to the confirmation
of ISA. Whilst the FHI were following current legislation and current and established
policy practices additional information was also passed to any company operating a
site where ISA was confirmed. This information included; the criteria for confirmation
of the disease referencing both the OIE and any relevant legislation; the
observations made by FHI on-site; the observations made by FHI at post-mortem
and the results of any laboratory tests. This information was sent out by e-mail and
followed with a hard copy by post on the day of confirmation.
59
The working practices of the FHI have now been updated, so in any future outbreak,
this information will be supplied as a matter of course.
Appendix 4 Terms and acronyms used in this report
Terms and acronyms relating to the virus
ISA Infectious salmon anaemia disease
ISAV Infectious salmon anaemia virus (not necessarily causing ISA)
HPR0 Putative ancestor ISAV, believed to be avirulent
2008-0982 Virus causing 2008/9 outbreak in Shetland
1998-390 Virus causing most of 1998/9 outbreak in Scotland
Terms and acronyms relating to diagnostic methods
TO cell A cell line originally cultured from salmon head kidney
leucocytes used to replicate ISAV
IFAT Indirect fluorescent antibody test (a diagnostic test)
qPCR quantitative Real-Time Polymerase Chain Reaction (a
diagnostic test)
Se Test Sensitivity: probability that if infection is present test
detect it
Sp Test Specificity: probability that a negative sample will not test
positive
Pooling Taking tissues from multiple fish to reduce diagnostic costs
Other terms acronyms
MSS Marine Scotland Science
FHI Fish Health Inspectors from MSS
MA Management Area as defined by MSS
LPP Lerwick Processing Plant processing plant
SPP Scalloway Processing Plant
‘bus stop’ The practice of collecting fish from multiple farms to take on to
a processing plant or other destination
Synchronous
fallowing
Fallowing all the sites in a defined area (ideally an MA) at the
same time
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© Crown Copyright Year 2010
Marine Scotland – Science
Marine Laboratory
375 Victoria Road
Aberdeen
AB11 9DB
Copies of this report are available from the Marine Scotland website at
www.scotland.gov.uk/marinescotland
61
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Short-term (48 h) exposure of healthy Atlantic salmon Salmo salar L. smelts to infectious salmon anemia (ISA)-inoculated cohort smelts showed that the disease was transmitted with near 100% mortality from Day 7 post-inoculation and onwards. This is more than a week before the inoculated fish show any clinical signs and long before the typical petechial bleedings occur. A bloodborne transmission of the disease is therefore unlikely. Skin mucus, faeces, urine and blood, isolated from ISA-inoculated smelt, transmitted the disease to healthy cohort smelt with variable efficiency depending on how the inoculum was administered. All the sources were infectious and transmitted the disease with high efficiency when injected intraperitoneally (i.p.) into cohort smelt. After i.p. injection, skin mucus had somewhat lower infectivity than blood homogenates. Furthermore, in some experiments application of skin mucus to the gills was as efficient as i.p. injection for transmission of the disease. When introduced into the stomach none of the inocula caused ISA. Coprophagy thus seems to be ineffective in the transmission of TSA under laboratory conditions. Skin mucus from non-inoculated cohabitants exposed to ISA-inoculated smelts for 2 d transmitted the disease with close to 100% efficiency to healthy cohort smelts when injected i.p. This indicates that the infectious agent is waterborne and absorbed by the skin mucus rather than being secreted with the skin mucus. Since healthy smelts have an intact skin barrier, proximity to inoculation directly to the vascular bed seems unlikely. An ultrastructural study of 10 different organs, all in close proximity to the secretions/excretions, revealed that at early stages of the disease, the virus was exclusively found in the pillar cells and endocardial cells. This indicates that the gills are the most Likely port of entry of the virus. It also supports a causal relation between the observed virus and the disease.
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The emergence of infectious salmon anaemia virus (ISAV) in several countries outside Norway and frequent new outbreaks of the disease within Norway, strongly suggests that there are natural reservoirs for the virus, probably in fish occurring in the coastal waters. Both in Norway and Canada fish farmers have claimed that there could be a possible connection between wild herring (Chipea harengus) migrating through fish farms and an outbreak of ISA in the same farms. It has also been claimed that wet feed made from herring could contain the ISA virus and, hence, transmit the disease to salmon (Salmo salar). Both these claims are "mythical" in that respect that they are not based on any scientific study or verification that the ISA virus may propagate in herring. Hence, the aim of this study was to challenge herring with the ISA virus, check for virus replication and see if the virus could be transmitted from challenged herring to disease-free Atlantic salmon. With the help of RT-PCR it was shown that the herring became infected with the ISA virus after bath challenge. A drop in haematocrit towards day 20 followed the infection. One salmon that was challenged with filtered homogenate made from ISA challenged herring died. However, most of the salmon survived, but they were positive in the RT-PCR test. It is concluded that the ISA virus is able to propagate in herring and that the herring may be an asymptomatic carrier of the virus.
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