Aquaculture 324–325 (2012) 1–13

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Review

Epidemiology of Renibacterium salmoninarum in Scotland and the potential forcompartmentalised management of salmon and trout farming areas

Alexander G. Murray a,⁎, Lorna A. Munro a, I. Stuart Wallace a, Charles E.T. Allan a,Edmund J. Peeler b, Mark A. Thrush b

a Marine Scotland Science, Marine Laboratory, Aberdeen, UKb Centre for Environment, Fisheries and Aquaculture Science, Weymouth, UK

⁎ Corresponding author at: Marine Scotland Marine LE-mail address: sandy.murray@scotland.gsi.ac.uk (A

0044-8486/$ – see front matter. Crown Copyright © 20doi:10.1016/j.aquaculture.2011.09.034

a b s t r a c t

a r t i c l e i n f o

Article history:Received 18 November 2010Received in revised form 20 September 2011Accepted 26 September 2011Available online 1 October 2011

Keywords:BKDRenibacterium salmoninarumAquacultureSalmonidsCompartmentalisationScotland

Bacterial kidney disease (BKD) (caused by Renibacterium salmoninarum) can result in significant mortality inScottish salmon farms, but is considered to be a minor issue on trout farms. Controlling R. salmoninarum in-fection in trout to protect farmed salmon would be effective only if the risk posed from trout is significantboth in absolute terms and relative to other potential sources of R. salmoninarum. To assess this, three com-plementary reviews are undertaken: review of data quality on BKD in Scotland and the national level preva-lence and dynamics these data imply; case studies of recent BKD outbreaks in Scotland; and an assessment ofthe epidemiological and management factors that maintain and spread R. salmoninarum within and betweenthe trout and salmon industries. These are then synthesised into a conclusion on the factors required for con-trol of BKD in salmon. Most observed spread of R. salmoninarum occurred within single species or even com-panies, so the majority of cases in farmed salmon are linked to other salmon (and not to trout) farms. There issubstantive geographical separation of areas of production for trout and salmon and transmission betweensalmon and trout networks is limited. The bacterium does not survive long in water so hydrodynamic trans-mission is likely to be localised. Currently R. salmoninarum is extremely rare in Scottish wild fish; this has notalways been the case. Wild fish therefore probably play a limited role, but might act as reservoirs or vectors.The general conclusion is that to a large extent the transmission of R. salmoninarum in salmon and trout pro-duction can be separated and so there is potential to compartmentalise BKD controls, either by host species orgeographical area.

Crown Copyright © 2011 Published by Elsevier B.V. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22. The prevalence and dynamics of BKD and R. salmoninarum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

2.1. Sampling and diagnostic methods used for the surveillance of BKD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32.2. Prevalence and persistence of BKD estimated from official surveillance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

3. Case histories of BKD in Scotland . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43.1. Persistent infection in table-production rainbow trout farms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43.2. Dynamic outbreak from a trout hatchery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43.3. Dynamic outbreaks in marine salmon on the west coast . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43.4. BKD in marine salmon from Shetland . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63.5. An outbreak in marine trout linking salmon and trout? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

4. Epidemiological factors behind the dynamics of BKD in Scotland . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74.1. Management and eradication of BKD and R. salmoninarum on farms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74.2. Vertical transmission . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84.3. Geographical structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94.4. Fish movements network structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94.5. Other anthropogenic networks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104.6. Hydrodynamic transport . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

aboratory, 375 Victoria Road, Aberdeen, AB11 9DB, Scotland, UK..G. Murray).

11 Published by Elsevier B.V. All rights reserved.

2 A.G. Murray et al. / Aquaculture 324–325 (2012) 1–13

4.7. Wild and escaped fish . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105. Synthesis and conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

1. Introduction

Infection with Renibacterium salmoninarum can cause bacterial kid-ney disease (BKD) in a variety of salmonid species in North America,western Europe, Chile and Japan (Austin and Austin, 2007; Toranzo etal., 2005); BKD has recently also been reported from Turkey (Savas etal., 2006). Bacterial kidney disease was notifiable to the World Organi-sation for Animal Health (O.I.E., 2006), but this status ceased in 2006,however it remains legally notifiable in the United Kingdom (UK), in-cluding Scotland.

R. salmoninarum can be transmitted vertically (Evelyn et al., 1986)and horizontally, particularly by the faecal-oral route (Balfry et al.,1996). However the bacterium survives poorly in the aquatic envi-ronment, so longer-distance transmission is likely to be associatedwith movement of fish (Austin and Rayment, 1985) or other anthro-pogenic routes. Evaluation of the significance of these potential routesof transmission as they apply in Scotland is a major component of thisreview (Section 4).

In Scotland BKD occurs in Atlantic salmon (Salmo salar) and rain-bow trout (Oncorhynchus mykiss) farms (Bruno, 1986, 2004). Histori-cally it occurred in wild Atlantic salmon as far back as the 1930s(Mackie et al., 1933; Smith, 1964) but BKD has not been reportedfrom wild Scottish fish since the 1960s, recent evidence of R. salmoni-narum in wild fish is reported in this document. Impacts of BKD onfarmed trout are considered less economically serious than on farmedsalmon where higher levels of mortality can occur in valuable marketsize fish. This disease is a significant contributor to the total mortalityof farmed salmon in Scotland although it is less significant than dis-eases such as pancreas disease and infectious pancreatic necrosis(Soares et al., 2011). Relatively few salmon farms have BKD so theloss-per case can be substantial and there is a potential for the num-ber of infected farms to increase substantially. It is therefore desirableto minimise the exposure of farmed salmon to R. salmoninarum, butcontrol of infection in trout may be less cost effective. It may be pos-sible to create separate compartments (Zepeda et al., 2008) withinwhich to manage infection in salmon and trout independently, butthe practicality of this depends on the degree of interaction betweenfarmed trout and salmon.

Bacterial kidney disease has been managed in Scotland using asystem of Designated Area Orders (DAOs) imposed on farms thathave been confirmed to be infected with R. salmoninarum and importrestriction under an EU approved Additional Guarantees (AG)

0

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2002 2003 2004 2005 2006 2007 2008 2009

Date

% o

f site

s

Fig. 1. Prevalence of BKD in terms of percentage of farms with DAOs in place over the pe-riods January 2002–June 2008. Thin solid line=trout farms, thin dashed line=salmonfarms, thick solid line=both salmon and trout farms.

programme (Munro, 2007). The DAOs imposed movement restric-tions, prohibiting the movement of fish from infected farms to otherfarms with the exception of those of similar health status. These re-strictions are now called Confirmed Designation Notices (CDNs)under the Aquatic Animal Health (Scotland) Regulations 2009, butthe term DAO is used here as it applied over the period analysedwithin this paper. The AG programme allowed the UK to ban importsof ova and fish from areas with BKD, but as a requirement had to havea BKD eradication programme (2006/88/EC). This programme wasachieved using the movement restrictions of DAOs and a programmeof fallowing affected populations.

However, the controls caused problems with costs falling dispro-portionately on trout farmers and the benefits going to the salmonfarmers. Furthermore the UK abandoned the AG programme forBKD (EU, 2010) as progress towards eradication could not be con-firmed (Fig. 1). Scotland therefore needed to develop new controlson BKD. In order to assess the potential for optimising control ofBKD in salmon without imposing unnecessary costs on the trout sec-tor we review the epidemiology of transmission of R. salmoninaruminfection to farmed salmon. Transmission to salmon farms mightoccur from activity within the salmon industry, or from a wild fishreservoir, or it might occur from trout farms (Murray et al., 2011). Ifthe risk from trout is not significant in both absolute terms and rela-tive to infection risk from the other two sources then reduction of thetrout-associated risk would not significantly protect salmon. Futurechanges in the prevalence of R. salmoninarum in the trout farms orthe structure of the trout industry could change the risk posed bytrout.

This review consists of three parts: (1) a review of the quality ofdata obtained from surveillance for BKD and R. salmoninarum in Scot-land and an overview of BKD prevalence and dynamics at the nationalscale obtained from this; (2) detailed case studies of recent outbreaksin BKD and R. salmoninarum infection in Scotland to identify patternswithin the outbreaks; (3) a review of the epidemiological processesand management practices that control these observed distributionof BKD in Scotland. We then combine these reviews to develop (4)a synthesis and conclusion as to the potential for control of BKD inScotland that minimise impact in salmon while imposing minimalcontrols on trout to achieve that end.

The interactions identified for the spread of R. salmoninarum canbe generalised into a template for assessment of the potential forcompartmentalised management in multi-species or multi-sectoraquaculture industries, adapted for local industry network and theepidemiology of the relevant pathogen. Due to the large number ofspecies produced in aquaculture worldwide the issue of identificationof suitable compartments for disease management is likely to be ofincreasing importance.

2. The prevalence and dynamics of BKD and R. salmoninarum

In order to assess the potential for control of BKD in salmon farmswe need to understand the quality of our knowledge on the distribu-tion and dynamics of BKD and R. salmoninarum in Scotland. To do this,we review the surveillance methods that are used for the official im-position of DAOs for BKD. We use the imposition and removal of theseDAOs to evaluate the prevalence and dynamics of BKD at the nationallevel.

3A.G. Murray et al. / Aquaculture 324–325 (2012) 1–13

2.1. Sampling and diagnostic methods used for the surveillance of BKD

Implementation of the DAO-based control measures on farmsdepended on the detection and confirmation of the disease (BKD)or pathogen (R. salmoninarum). Scotland's official Fish Health Inspec-tors (FHI) therefore systematically collected data on these. Our epide-miological understanding of the prevalence of R. salmoninarum is,historically, entirely dependent on this official sampling since therehas been no other screening (e.g. for research) of farms that are notalready known to be infected, although research screening of wildfish has occurred. It is therefore upon the quality of the official datathat any systematic understanding of BKD dynamics in Scotlandmust be based.

Detection of clinical BKD can be relatively straightforward as thepresence of disease is apparent (see Richards (2011) for official listof symptoms) and, being notifiable, any suspicions of its presencemust be reported to FHI who will take kidney samples from 150 fishgrouped in pools of 5. Diagnostic tests using enzyme-linkedimmuno-sorbent assay (ELISA) followed by bacterial culture are sen-sitive for confirming the presence of R. salmoninarum from clinicallydiseased fish (Bruno et al., 2007). So this surveillance that is passivein its first steps is likely to lead to official confirmation at least inthe presence of significant levels of BKD.

Detecting sub-clinical R. salmoninarum infection can be problem-atic. If there is no disease to observe then passive surveillance cannotwork. Active surveillance programmes have limited ability to targetdiagnostic testing on individuals if they are asymptomatic, althoughseasonal sampling may help as infection is more likely to be detectedin autumn or early winter (Austin and Austin, 2007). In Scotland in-spections undertaken by the FHI are focused when water tempera-tures of around 12 °C are expected. Even if an infected population issampled the proportion of fish infected within that population maybe low and within infected individuals the bacteria may persist asonly a few isolated clumps in the kidney (Austin and Rayment,1985). Therefore, even if infected populations are sampled there is agood chance that material from infected individuals will not be in-cluded in the sample and diagnostic test sensitivity may be low(Hall et al., 2011).

Regular background surveillance consisted of FHI taking head kid-ney samples from 30 fish, in pools of five, from all farms on a biennialbasis until 2010. These were screened using ELISA. If ELISA resultswere positive a confirmatory sample of 150 fish were sampled. His-torically this confirmatory sample has been diagnosed using cultureon Mueller Hinton Agar plus L-cysteine hydrochloride and antibiotics(MHCA) plates but, since 2005, quantitative real-time polymerasechain reaction (qPCR) has also been an approved method for confir-mation. Culture can take up to 12 weeks to confirm and the absenceof R. salmoninarum cannot be assumed until the 12 week test periodwas complete. Diagnostic test methodologies are detailed by Brunoet al. (2007). Both culture and qPCR were acceptable confirmatorytests listed by the OIE when BKD was still internationally notifiable(O.I.E., 2006); the OIE no longer requires notification of BKD. Thislevel of screening is likely to be very insensitive for the detection ofsub-clinical infection as prevalence is often low and it appears ELISAis not particularly sensitive especially when samples are pooled(Hall et al., 2011). However, this was the screening protocol laiddown by the EU for assurance of disease freedom (EU, 1999) and soit was used for its approved purpose in Scotland.

Since the summer of 2010 testing has been undertaken only if thereis suspicion of clinical BKD, i.e. screening for subclinical infection hasceased. It is believed that this change will have made very little differ-ence to the probability of detection (Hall et al., 2011), however weonly use data from before this date to ensure consistency.

Farms holding broodstock were sampled at the same level as otherfarms, i.e. 30 fish every second year using ELISA diagnostic testing ofkidney samples from pools of 5 fish. This means sampling was unlikely

to pick up sub-clinical R. salmoninarum (Hall et al., 2011), however ver-tical transmission ismuchmore likely to occurwhen infection is clinicaland since BKD is notifiable such diseased fishwould bemore likely to bedetected. Broodstock farms exporting to Ireland or Chile were tested atthe 150 fish level on an annual basis (ELISA on kidney). Occasionally,wild broodstock fish that were kept alive for stripping were tested forR. salmoninarum using ovarian fluid samples.

To confirm eradication of R. salmoninarum from a farm subsequentto fallowing, pooled kidney samples from 150 fish at six monthly in-tervals over a two year period had to screen negative by ELISA.

Wild fish have also been screened by the FHI and research scien-tists, effort has varied over time. Diagnostic testing was by bacterialculture (or sometimes ELISA) using head kidney, or whole fish if toosmall for kidney sampling. A total of 4539 wild fish were screenedover 1989–2004. After 2004 qPCR screening was used, 1435 sampleswere taken over 2005–7 (Section 4.7).

Detailed data on mortality and its causes are collected by compa-nies involved in fish farming (Soares et al., 2011). The mortality re-cords have been available from a company that was affected by asubstantial number of the cases of BKD in salmon in the period2003–7. These data have been used to analyse patterns in the mortal-ity ascribed to BKD by farm managers of marine farmed salmon.

Marine Laboratory researchers have also followed outbreaks in de-tail, using the more sensitive qPCR for diagnostic purposes. This hasallowed the outbreaks and on-farm spread to be followed (Wallace etal., 2011), but researchers have not looked for R. salmoninarum on unde-signated farms.

In conclusion, surveillance for clinical BKD was likely to be reason-ably good, with clinical cases of notifiable disease being reported anddiagnostic methods being highly sensitive and specific for clinical dis-ease. However, data on sub-clinical infection are very limited, with di-agnostic methods being of low sensitivity and surveillance limited toactive biennial visits. Therefore the prevalence and distribution of R.salmoninarum in Scotland is highly uncertain.

2.2. Prevalence and persistence of BKD estimated from official surveillance

The number of DAOs that were in force in each month has beencalculated for the period January 2002–June 2008 using the net num-bers of DAOs issued and revoked. This has been divided by the num-ber of farms in production in the particular year (Walker, 2010) togive the proportion of fish farms that were known to be infected.From this we can see that infection levels have been consistently atabout 2.5% of farms over this period (Fig. 1). However, DAOs have ap-plied to approximately 15–20% of trout farms and 0–1% of salmonfarms with periods of absence of any DAOs on salmon. Since around90% of farms rear salmon the industry average 2.5% is much closerto prevalence in salmon than in trout.

The potential for subclinical infection means that absence of DAOsdoes not mean an absence of R. salmoninarum, and so DAOs are likelyto underestimate prevalence of infection. However, trends in num-bers of DAOs are likely to be a useful measure of the relative preva-lence of infection. Assuming onset of disease, and hence detection,has not changed substantially, the proportion of infected farmsexpressing BKD, and the therefore the proportion which are detected,should remain reasonably constant.

The prevalence of farms with DAOs showed little sign of declininghence the eradication policy could not be demonstrated to be work-ing. Since this was a requirement of the AG programme (Munro,2007) these have ceased for the UK, which is no longer listed as aBKD free territory (EU, 2010). Eradication from salmon may be possi-ble if these can be isolated from trout, as there have been periods withan absence of orders.

The time between imposition and lifting of DAOs from farms canbe used to give information on the transience or persistence of infec-tion (Fig. 2). Persistence of infection on farms varies depending on the

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cageSalmon Trout cage Trout fishery Trout sea Trout tanks Trou ponds

farm type

aver

age

time

(yea

rs)

sinc

e D

AO

im

pose

d(13)

(6)

(1) (3)

(2) (3)

Fig. 2. Persistence of infection with R. salmoninarum (in years) in different types of fish production, as measured between the initial imposition of Designated Area Orders (DAO),and time revoked (or until 1st June 2008 if still in force at that time). The numbers in brackets indicate the number of DAOs imposed.

4 A.G. Murray et al. / Aquaculture 324–325 (2012) 1–13

type of farm. For trout held in tanks, and for salmon farms, infection isdynamic with infection-free status being regained after a relativelyshort period of months to at most 2 years. For trout in some freshwa-ter cage farm infection has persisted for decades due to the practice ofcontinuous stocking (Wallace et al., 2011).

From 1960 until 2003 neither BKD nor R. salmoninarum wasreported in wild Scottish fish. In 2003 an ELISA positive screening re-sult was obtained from herring (Clupea harengus) obtained from acage with infected farmed salmon (unpublished data) and in 2005and 2007 qPCR evidence of R. salmoninarum was obtained from wildfish in the vicinity of infected trout farms (Wallace et al., 2011). Thebacterium has also been cultured from wild English fish in a recentsurvey (Chambers et al., 2008). We examine the information onwild fish in more detail later when we consider their potential as res-ervoirs or vectors (Section 4.7), however here we can state the prev-alence of infection was very low to negligible and BKD itself has notbeen detected in Scottish wild fish for half a century.

3. Case histories of BKD in Scotland

Scottish BKD outbreaks from the 1990s–2010 are analysed as casestudies on the spread of BKD. The data used for these case studieswere collected by FHI and researchers from the Marine Scotland Ma-rine Laboratory, Aberdeen and the aquaculture industry. Outbreaks ofBKD in the last two decades have occurred in Scotland in freshwaterand marine rainbow trout and marine salmon farms (Bland, 2007;Bruno, 2004; Murray et al., 2011). The characteristics of these out-breaks have varied considerably depending on the type of farminvolved.

We categorise the epidemiological patterns in recent Scottish BKDcases as: 1. Persistent cases in freshwater trout farms; 2. Outbreaks infreshwater trout farms linked to a hatchery in 2005; 3. Outbreaks inmarine salmon farms on the west coast of Scotland; 4. Outbreaks inmarine salmon farms in Shetland; and 5. Outbreaks in marine troutand salmon farms that are possibly linked. More detailed descriptionsof these outbreaks are provided in Murray et al. (2011).

3.1. Persistent infection in table-production rainbow trout farms

Several trout farms in Scotland have been infected with R. salmoni-narum, sometimes leading to clinical BKD, over many years (Fig. 2).Four of these farms have been continuously under DAOs since 1981 or1982. These are freshwater cage farms where continuous stocking waspractised and therefore they were never fallowed. Recently this man-agement practice has changed with some fallowing being introduced(Wallace et al., 2011). It is likely that the source of infection to naïve co-horts placed on farms was by horizontal transmission from establishedstocks on the farm, although it is possible that some cohorts could have

been infected before stocking and this seems a likely source of reintro-duction following farm-level fallowing (Wallace et al., 2011). There isno evidence of these farms infecting other farms and all are consideredas dead-end nodes in the farm network (fish not moved off except forharvesting, although escapes are possible; see Section 4.4).

As these persistently infected farms do not appear to spread infec-tion to other farms, this suggest that R. salmoninarum positive farmscan be effectively isolated if there is no off-site movement of fish.However it is likely that the local spread of R. salmoninarum occursfreely between cages and escaped fish may carry infection.

3.2. Dynamic outbreak from a trout hatchery

In 2005 BKD was confirmed (2/150 fish by culture) at a freshwatertrout farm in the Scottish Highlands. This infection was traced to thesupplying hatchery in central Scotland. Contact tracing showed re-cent fish movements to seven farms (Bland, 2007), two other Scottishfarms (1/150 and 3/150 by culture) and one English farm were con-firmed R. salmoninarum positive. One Scottish farm was placedunder suspicion due to an ELISA positive result (1/91) but was notconfirmed as culture tests were negative (0/150 fish). Full diagnosticresults are available in Bland (2007).

The hatchery was cleared of R. salmoninarum by fallowing on atank-by-tank basis; this approach was effective as this was an indoorfacility with good internal biosecurity which could be maintained. Asa result the DAO was revoked in November 2005. One of the Scottishfarms was fallowed and the infection cleared, while the other was fal-lowed first on a pond-by-pond basis and infection was either not re-moved or recurred. Subsequently this farm fallowed at the farm leveland infection was removed.

The spread of the epidemic reflected the network of movements offish transported between farms (see Section 4.4) and cases were spa-tially separated (see Section 4.3). This outbreak illustrates the capac-ity of highly connected nodes in a farm fish movement network tospread R. salmoninarum (and potentially other pathogens) betweenindividual farms and across political boundaries.

3.3. Dynamic outbreaks in marine salmon on the west coast

An outbreak affecting six west coast salmon farms (WS1–WS6)occurred in 2003 (Fig. 6; Table 1). Five of these sites (WS1–WS4and WS6) returned culture positive results (Table 1). The site WS5did not test positive by culture, but it did test positive by ELISA. Thecompany ascribed 13.5 tonnes of losses on this farm to BKD, so al-though it officially only had a temporary thirty day notice (TDN) im-posed we treated this site as BKD positive for epidemiologicalanalysis. During this outbreak a herring (one of nine) taken from

Table 1Detection of R. salmoninarum from salmon farms on Scottish west coast 2003–7. The ELISA results refer to individual fish except WS7 and WS10 which are for pools of five. Des-ignated Area Orders were imposed, except on WS5 and WS7 where only TDNs were imposed. Mortality was ascribed to BKD by the company using data collected from 2001 to2008.

Farm Date Culture ELISA Mortality(tonnes)

Comment

WS1 Apr 2003 1/2 116/155 20.3 Also 1/9 herringWS2 May 2003 1/3 1/3 0.1WS2 Mar 2007 32/51 8/11 70.9WS3 Jun 2003 2/3 3/150 11.4WS4 Jun 2003 5/6 6/150 133.3 Also positive in November 2003WS5 Jun 2003 6/149 13.5 TDN onlyWS6 May 2003 1/1 1/1 75.7 Also positive June 2004WS7 Dec 2004 1/1 0 Also histology positive in March

2005. TDN onlyWS8 Apr 2005 1/2 1/2 6.8 Also positive in May 2005WS9 0 DAO imposed because fish

received from WS10WS10 Apr 2007 1/150 2/30 0

5A.G. Murray et al. / Aquaculture 324–325 (2012) 1–13

within a cage on farm WS1 in April 2003 tested positive by ELISA, afurther 22 herring sampled in May tested negative.

Further positive diagnostic results were obtained in 2005(Table 1) with WS7 only ELISA positive, while WS8 and WS2 (forthe second time) were confirmed positive. Another farm WS8 wasin the western isles, outside the area of the earlier outbreaks(Fig. 3), indicating the geographically widespread nature of the BKDcases. All DAOs from this and the previous outbreak were revokedby mid 2006 (Fig. 3). In 2007 site WS10 was confirmed, with WS2reconfirmed for the third time, by culture. FarmWS9 did not test pos-itive but had a DAO placed on it because fish were moved onto it fromWS10. These farms lie to the north of the area covered by the map(Fig. 3). None of the farms listed here were among the five salmonfarms affected by BKD between 1976 and 1985 (Bruno, 1986).

Data on the mortality ascribed to BKD, and for tracing sources ofsmolts, were available for 2004 to 2007. Mortality varied substantiallybetween farms, although they appear to have been infected at thesame time (Fig. 4). Some farms infected with R. salmoninarum had

Fig. 3. Location of the Loch Sunart, Loch Linnhe (Section 3.3) and Loch Etive (3.5) clustered odrains into Loch Etive; FS1 is a putatively infected freshwater salmon farm (3.3) (located offment Areas (MA).

no mortality ascribed to BKD, while others were severely affectedand losses varied from 0 to 133 tonnes per farmwith 83% of mortalityaccounted for by three farms (WS2, WS4 and WS6). These losseswere divided by biomass, obtained using records of biomass collatedmonthly by the Scottish Environmental Protection Agency (SEPA), toshow that the mean of the loss ascribed to BKD for these farms was3.3%, and the worst affected lost 12.6%, of maximum biomass.

These outbreaks only affected one company, suggesting anthropo-genic spread. Outbreaks were temporally and spatially clustered, withthree simultaneous cases in Loch Sunart, however BKD was alsodetected on Skye as part of the same outbreak; and a neighbouringfarm within Loch Sunart remained uninfected. Later cases wereeven more widespread (WS8, and WS10) While it is possible thatfarm-to-farm spread had occurred within the clusters, anthropogenicspread seems the most likely explanation for this pattern.

Generally BKD did not recur on salmon farms subsequent to fal-lowing (hence short duration of BKD DAOs in salmon, Figs. 2 and 4).The farm WS2 is an unusual case being R. salmoninarum positive in

utbreaks of BKD. FT2 is the location of a persistently infected freshwater trout farm thatthe top right of the map); black circles represent towns and numbers refer to Manage-

WS1

WS2

WS8

WS3

WS4

WS5

WS6

WS7

2004 2005 2006 20072003

3-30 30-300 300-3k 3k-30k 30k-300k

Linn

he c

lust

erS

unar

t clu

ster

Skye

Western Isles

WS9

WS10

nort

hwes

t

Fig. 4. Outbreaks of BKD on west coast salmon farms 2003–7. Symbols represent:=quarterly mortality attributed to BKD in industry records (symbol size is pro-

portional to level of mortality);▲=DAOs imposed ( =TDN only);▽=DAOsrevoked.

6 A.G. Murray et al. / Aquaculture 324–325 (2012) 1–13

three consecutive production cycles. In the first a DAO was imposed,but no mortality was attributed to BKD, limited mortality occurredin the second production cycle and the DAO was re-imposed in asso-ciation with substantial BKD attributed mortality in the third produc-tion cycle. This farm is close to a processing plant, so there is apossibility that it might have been exposed to infected material han-dled at that plant depending on the biosecurity in place at the time.

The most significant association with all the BKD outbreaks is theuse of smolts from specific freshwater farms. The 2003–5 outbreaksshowed very significant links to two freshwater smolt producingfarms FS1 and FS2 (not shown), both through contact tracing andanalysis of numbers of movements onto the farms. Overall the indus-try database records 34 deliveries of smolts to sites that developedBKD and 658 deliveries to farms that did not, giving odds of 34:658of a delivery being associated with BKD (many sites received multiplesmolt deliveries). However, farms receiving smolts from FS1 and FS2had higher odds for developing BKD, 19:94 and 10:43 respectively.These give highly significant Odds Ratio (Kelsey et al., 1996) of BKDin farms receiving smolts from FS1 and FS2 of 3.9 (p=2.9E−5) and4.5 (p=0.00054) respectively, relative to averaged odds for thewhole database using the Fisher's Exact test. It therefore seems likelythat there was a link to infection via fresh water farms.

Although the freshwater farms FS1 and FS2 are linked epidemio-logically with cases of BKD, they never tested positive. It is possiblethat a low-level undetected sub-clinical infection may have existedon these farms. The weakness of surveillance for subclinical infection

Table 2Detection of R. salmoninarum from salmon farms in Shetland 2006–2009. Result refers to ELindividuals were analysed using light microscopy.

Farm Result Restriction Dateimp

SS1 1/1 TDN 28/0SS2 1/6 TDN 26/0SS3 6/7 DAO 30/0SS4 4/38 DAO

(17/07/08)27/0

SS5 2/62/32

Fallowed 04/012/0

SS6 1/11* Fallowed 14/0

(Hall et al., 2011) means that a lack of detection of any such infectionwould not be at all unlikely. Farm FS1 was potentially exposed to aneighbouring trout farm (again negative but a possible link to thetrout network), to wild char (Salvinus alpinus) (a species known tocarry R. salmoninarum in other countries (Jonsdottir et al., 1998) al-though data from Scotland are lacking), or to migratory salmonidspossibly transporting infection from the marine salmon farms inLoch Linnhe. It is also possible that the fish could have been infectedprior to stocking to these freshwater farms. Any of the risk routescould have been the source of infection to FS1.

3.4. BKD in marine salmon from Shetland

In recent years R. salmoninarum has been detected by ELISA on sixfarms throughout the Shetland Islands although only two have beenconfirmed by culture (Table 2). All six farms affected were ownedby company Z at the time R. salmoninarum was detected, althoughownership of some farms has since changed. Farms SS1 and SS2were positive by ELISA but were not confirmed by culture (Table 2);although this is insufficient to impose DAOs we considered ELISAgood evidence that these farms had been exposed to R. salmoninaruminfection (Hall et al., 2011). The affected farms were in different re-gions of Shetland, and farms belonging to other companies in theseregions were not affected. The affected fish from these differentfarms were all sourced from different freshwater smolt farmsthroughout Scotland. Farms SS5 and SS6 generated positive test re-sults but were fallowed before confirmatory testing occurred(Table 2). The R. salmoninarum positive from SS6 was obtained as aresult of pathogen screening during an outbreak of infectious salmonanaemia (ISA) and the farm was depopulated due to confirmation ofISA disease. Another farm, belonging to the same company, had aDAO imposed in May 2008 because of epidemiological links to aninfected farm, but the fish tested negative for R. salmoninarum sothis farm was not included in the analysis.

There is a significant association between Scottish farms belongingto the company and them becoming infected with R. salmoninarumsometimes leading to BKD. With a total of 107 farms in Shetlandand with 31 farms belonging to company Z then the probabilityusing the Fisher's exact test of all six of the positive farms belongingto company Z is p=0.0012, i.e. ownership is significantly associatedwith infection.

With no geographical association, so no evidence for hydrody-namic or other environmental contact, on the one hand, and a lackof common smolt origin on the other, the intra-company associa-tion strongly suggests anthropogenic spread of infection. This pat-tern of spread is different to the pattern observed for infectioussalmon anaemia virus (ISAV) in Shetland which was transmittedthrough the environment, probably hydrodynamically, and showedlittle or no relationship with company ownership (Murray et al.,2010). Intra-company spread of R. salmoninarummight be via a pro-cessing plant, although other companies used this plant and their

ISA screening expressed as positive pools/total number of pools except last case* where

restrictionosed

Smolt source Management area

6/06 4a6/07 Highlands 3a1/08 Dumfries 2c5/08 Argyll 3a

6/086/08

Jura 3a

1/09 3a

7A.G. Murray et al. / Aquaculture 324–325 (2012) 1–13

fish did not develop BKD. The use of the processing plant might beconfounded with other unmeasured company-based managementpractices (e.g. shared personnel, equipment etc.). A total of 31farms from company Z used this processing plant and in contrast24 farms from eight other companies used this plant and had nopositive farms. Although this is statistically consistent with contactwith the processing plant being the sole risk factor for infection as-sociation of risk with farm ownership is much stronger. In some in-stances well boats were used for ‘bus-stop’ deliveries (picking upfish from a number of farms on the same boat journey to the proces-sing plant (Murray et al., 2010)). These ‘bus stop’ harvests are morelikely to result in contact within a company than contact via a pro-cessing plant, which is shared by several companies.

3.5. An outbreak in marine trout linking salmon and trout?

Marine rainbow trout farms in Loch Etive (Fig. 3) have a historyof R. salmoninarum infection with DAOs imposed on two trout farmsin the loch in 2002. This sea loch is located near the outfall of theRiver Awe which drains Loch Awe, the location of two persistentlyinfected freshwater rainbow trout farms (two of those describedin Section 3.1). It discharges into the lower reaches of the LochLinnhe system, within which there have been BKD outbreaks in ma-rine salmon farms (Section 3.3). The five trout farms in Loch Etivehave been supplied with rainbow trout from FT1 (Section 3.3)which has been suggested, but not shown, to play a role in the infec-tion of FS1 which in turn has shown a highly statistically significantlink to the outbreaks in Loch Linnhe. Loch Etive therefore might bean area in which BKD outbreaks in salmon and trout are linked.

In April 2009 suspicion of BKD was reported at two marine troutfarms operated by one company and in close geographical proxim-ity (Fig. 3). This was confirmed by culture in May 2009 at MT1, MT3and MT4 (Table 3), but not at MT5, while MT2 was fallow. In May2009 suspicion of BKD was reported from two Atlantic salmonfarms (AS1 and AS2) located near the mouth of Loch Etive. One ofthese (AS1) was confirmed, while the other (AS2) tested negative(Table 3). In April 2010 BKD was confirmed at the previously nega-tive trout farm MT5 and in November 2010 at the previously fallowMT2. Additionally, MT4 screened culture positive in April 2010.

Spread among the trout farms reflected inter farm anthropogen-ic activity. Fish were moved between MT3 and MT4 (both direc-tions) and from MT3 to MT1 in the preceding months. Farms MT1,MT3 and MT4 also share a shore base. The farms MT1 and MT3 arealso located within 1 km of each other so contact through the envi-ronment, perhaps with water movement, may be possible, althoughMT4 is more distant (3.3 km). It was therefore hardly surprisingthat R. salmoninarum was spread among these farms, potentiallyby any of these routes. The infection at AS1 at the same time is sug-gestive of a link between salmon and trout through the environ-ment, but coincidental infection is entirely possible.

Table 3Detection of R. salmoninarum from marine trout (MT) and marine Atlantic salmon (AS)farms in the Loch Etive area 2009–10.

Farm Date ELISA(pools of 5)

Culture qPCR Comment

MT1 May 2009 2/30 3/150 2/2MT2 Nov 2010 2/30 24/150 2/30 Fallow 2009MT3 May 2009 2/2 10/10 2/2MT4 May 2009 2/2 8/10 2/2MT4 Apr 2010 1/1 6/150 1/1MT5 May 2009 0/30 N/A N/A NegativeMT5 Apr 2010 1/6 2/2 1/1AS1 May 2009 1/30 1/1 1/1AS2 May 2009 0/30 0/5 N/A Negative

4. Epidemiological factors behind the dynamics of BKD in Scotland

Data on the epidemiological factors behind the prevalence of BKDin Scotland was reviewed, considering both the persistence of R. sal-moninarum on infected farms and its spread between farms. Thesefactors are the two components of R0, the rate of spread divided bythe rate of removal of infection, that defines the potential for controlof a disease (Reno, 1998); for eradication R0 must be less than 1. Dis-ease can thus be eradicated either by increasing removal rate or by re-ducing its spread. Spread includes vertical transmission through ovaor smolts (including ova imports) and horizontal transmission be-tween farms. Horizontal transmission depends on some form of con-tact between farms, and so we reviewed the geographical location offarms before reviewing potential contact process of: networks of fishmovements, other anthropogenic activity, hydrodynamic transmis-sion and the role of wild fish as vectors or reservoirs.

4.1. Management and eradication of BKD and R. salmoninarum on farms

Fallowing is used to remove R. salmoninarum from infected cages orfarms, however cage-level fallowing in loch-based farms proved inef-fective because of horizontal transmission between cages (Wallace etal., 2011) and infection persisted on some such farms for decades(Section 3.1). Fallowing a pond-based farm on a pond-by-pondbasis has also proved ineffective (Section 3.2), but a tank-basedhatchery with high levels of biosecurity was successfully fallowedon a tank-by-tank basis (Section 3.2). Fallowing at the farm-levelhas a mixed history, it appears to be successful on most salmonfarms as BKD mostly does not recur after fallowing (Sections 3.3and 3.4). For trout farms there has been post-fallowing recurrenceon some farms (Wallace et al., 2011), but other farms appear tohave been cleared by farm-level fallowing (Section 3.2). An environ-mental reservoir such as water or sediment is considered unlikely(Austin and Rayment, 1985) but low levels of infection have beenfound in wild and escaped fish near the farm (Wallace et al., 2011)so these may undermine the effectiveness of fallowing. Alternativelyinfected fish might have been re-introduced when the farm wasrestocked; only one batch needs to be infected due to the potentialfor horizontal transmission between cages (Wallace et al., 2011).This would imply a more careful sourcing of fish by increasing thesensitivity of screening or/and limiting the number of source farmsto reduce the likelihood of re-infection.

Vaccination for BKD was not practicable in Scotland under theeradication scheme as it is difficult to distinguish vaccinated fishfrom infected fish, and some fish in BKD vaccinated populationsremained infected (Griffiths et al., 1998). In any case BKD vaccines ap-pear fairly ineffective relative to vaccines for other bacterial diseases(Newman, 1993; Toranzo et al., 2005), although possible new devel-opments with live vaccination with a R. salmoninarum related bacte-rium, Arthrobacter davidanieli, may be effective in the future(Toranzo et al., 2005). Recent vaccination trials involving this bacteri-um showed significantly reduced mortality of salmon relative tosalmon in the same cage only vaccinated against other diseases dur-ing a BKD outbreak (Burnley et al., 2010). However, R. salmoninarumwas still present and culturable, if at a significantly lower prevalence(Burnley et al., 2010), so while the vaccine controlled mortality thevaccinated fish remained carriers. To date, vaccines have not been ap-proved for the control of BKD in Scotland.

The disease can be treated with antibiotics (Austin, 1985) al-though such treatment does not necessarily eradicate the bacteriumfrom the infected fish as the antibiotic may not always reach the tar-get bacterium within the host (Austin and Austin, 2007; Bruno andMunro, 1986). Antibiotics can be useful for treating broodstock(Brown et al., 1990; Lee and Evelyn, 1994), since the bacterium canbe vertically transmitted. Disease can also be controlled with dietarysupplements (Austin, 1985; Lall et al., 1985).

0

5000

10000

15000

20000

25000

30000

35000

40000

45000

50000

1995

1996

1997

1998

1999

2000

2001

2002

2003

2004

2005

2006

2007

2008

Date

Impo

rted

ova

USA

Australia

Norway

Iceland

EU

Fig. 5. Numbers and sources of Atlantic salmon ova imported into Scotland from out-side the UK (Walker, 2009).

8 A.G. Murray et al. / Aquaculture 324–325 (2012) 1–13

It is possible that R. salmoninarum infection could clear from apopulation without any specific management action. However R. sal-moninarum causes persistent chronic infection and can persist at lowprevalence for long periods (Wallace et al., 2011). The long-term per-sistence on freshwater cage-based sites that do not fallow at the farmlevel (Fig. 2) suggests that a natural clearance of infection from a con-tinuously stocked site without any intervention is likely to be a rareevent.

In summary, vaccines and treatments might help control BKD atthe farm level, but they do not eradicate R. salmoninarum and so thefarms remain potential infection sources. Fallowing is not effective

Fig. 6.Map of locations of freshwater trout (dark); salmon (moderate); and mixed (light) faassociated with a hatchery outbreak (Section 3.2). Inset map I is the Shetland outbreak, SectBKD; III. The main west coast outbreaks and FS1, Sections 3.3 and 3.5; IV. An area of persishared by salmon and trout and to date not associated with BKD. The dashed line representsland) and trout (south east) farming.

at eradicating R. salmoninarum if carried out at the cage level exceptin tank-based farms. At the farm level fallowing is usually effective(especially in salmon) and where infection does occur after farm-level fallowing then reintroduction of infection with the input offish may be an explanation, although reservoirs of infection in wildor escaped fish cannot be ruled out (Section 4.7).

4.2. Vertical transmission

R. salmoninarum can be vertically transmitted (Evelyn et al., 1986;Paterson et al., 1981) and so could be introduced with imported ova.The AG programme allowed the UK to restrict imports of ova andsmolts from areas where BKD was present. However substantial im-ports of salmon and trout ova occurred with some 700,000 to 30 mil-lion salmon ova are imported each year (Fig. 5). The number of theseimports has increased with some coming from countries such as Ice-land, Norway and the USA where R. salmoninarum is present. Infec-tion was reported in Atlantic salmon broodstock in Norway in 2008(Nilsen and Sunde, 2008) illustrating the possibility for vertical trans-mission within the salmon industry. Although imports are certified asR. salmoninarum free any failure in this process could risk the importof infection. In addition, 519,000 smolts were imported in 2008, butonly from R. salmoninarum free areas within the EU; these numbershave been falling (Walker, 2010).

In 2008 25 million trout ova were imported into Scotland, includ-ing 1.49 million from the US and imports also occur from Australiaand South Africa (although not in 2008) making trout ova importstruly global (Walker, 2010). Imports of trout ova have the potentialto increase the prevalence of disease agents and impede eradication,as shown for infectious pancreatic necrosis virus (IPNV) in Ireland,

rms. The symbol size is proportional to the number of contacts. Arrows mark the spreadion 3.4, circles mark: II. An area of mixed salmon and trout farming not associated withstent R. salmoninarum infection in rainbow trout, Sections 3.1 and 5. A drainage basinan arbitrary divide between areas of predominantly salmon (northwest including Shet-

9A.G. Murray et al. / Aquaculture 324–325 (2012) 1–13

(Ruane et al., 2009), thereby indirectly increasing the risk from trout.None of the available evidence is suggestive of the vertical transmis-sion of R. salmoninarum in Scotland whether from domestic orimported broodstock, but the route is a biologically demonstrablerisk.

4.3. Geographical structure

The location of trout and salmon farms in Scotland was mappedusing geographic information systems (GIS) software (ESRI (UK)Ltd.). There is an apparent spatial separation of the salmon andtrout industries, with the majority of trout farms in the south andeast, while the majority of salmon farms are in the west or the North-ern Isles (Fig. 6). However, there is some overlap with trout and salm-on farms sharing a number of small areas (I to V).

In total 27 Scottish catchments, consisting of main river and smallcoastal catchments, contain both farmed salmon and trout (2003 dataused to be consistent with the contact network analysis). This list in-cludes 17 catchments with single mixed species farms so the risk hereis associated with intra-farm as opposed to inter-farm interactions. Inaddition, some of these facilities are operated by fishery trusts or Dis-trict Salmon Fishery Boards so represent fish of wild origin, hencethese will not be dealt with in this section. Of the 10 remaining catch-ments, some of which have multiple salmon farms located within,there were 14 salmon farms which share a water course with troutfarms. Five of these salmon farms are separated from the nearesttrout farm by 5 km or less at 0.11, 0.34, 0.84, 4.1 and 5.0 km respec-tively. In 2003 there were 176 smolt production farms in operation(Walker, 2010), therefore the great majority of Scottish freshwatersalmon production occurs at farms that do not share catchmentswith trout farms.

Stocked trout fisheries are distributed throughout Scotland, how-ever most are located near large population centres, and thereforewithin the ‘mainly trout’ zone of south-eastern Scotland. Many fisher-ies are hydrographically isolated from fish farms but others may havepotential hydrodynamic contact e.g. fisheries in upstream reservoirs.The locations of these trout fisheries were not documented by the

Fig. 7. The network for movement of farmed trout in Scotland, and its connections to the salto the salmon farming network are indicated by thick arrows connecting a fish symbol (viatrout farms have no connections to the salmon network and two minor clusters have onlysalmoninarum infections (white) and Scottish farms involved in a hatchery based outbreak

FHI and so numbers within the ‘salmon’ region (Fig. 6), and numbersof movements where transport was between the regions, have notbeen quantified. Subsequently these fisheries are required to be reg-istered under the Aquatic Animal health (Scotland) regulations2009, so this knowledge gap is being addressed.

Transmission of R. salmoninarum between the trout and salmonsectors requires that the geographical separation be overcome. Inthe next sections we examine the routes of transmission that mightapply, these being anthropogenic movement of fish (Section 4.4)and other industry related contacts (Section 4.5) and transmissionthrough the environment with water movement (Section 4.6) or bywild/escaped fish (Section 4.7).

4.4. Fish movements network structure

A plot has been derived for the farmed fish movement network inScotland using data from transports authorised in 2003 in whichnodes (farms) were divided between salmon, trout and those produc-ing both (Green et al., 2009; Murray et al., 2011). This networkshowed that there were very few links between trout and salmonfarms and in addition the pure trout farms have few links to themixed species nodes.

There is wide interaction between salmon farms and the salmonnetwork (not shown) links most salmon farms together into a singlelarge network or epidemiological compartment (Green et al., 2009;Zepeda et al., 2008). This means disease may have the potential to be-come widespread with movements in this network, although thismay be less severe than it appears because of unidirectional move-ments and communities within the network which may localisespread (Green et al., 2009). There are a number of mixed species ele-ments within this salmon cluster but these are isolated from the maintrout farming network.

If farms producing trout only are plotted (Fig. 7) it can be seen thatmost form a network that is entirely separate from the salmon net-work. Two smaller sub-components connect into the salmon net-work, each by single contact routes, therefore the farmed troutmovement network is to a very large degree separate from the

mon farm network. Symbol size is proportional to the number of contacts. Connectionsa square mixed species farm). Note that the main trout cluster and an isolated pair ofone connection each to the network. Also shown are Scottish farms with persistent R.(black) (Section 3.2).

10 A.G. Murray et al. / Aquaculture 324–325 (2012) 1–13

salmon network. Outbreaks of BKD in Scottish trout have followedthe structure predicted by the network. Persistently infected farms(white symbols) which did not spread R. salmoninarum within thisnetwork are considered to be dead-end nodes (Section 3.1). Farmsthat were positive during the hatchery-based outbreak (black sym-bols) (Section 3.2) reflect movements from this hatchery. Thismatch between the network and outbreaks indicates that it is reason-able to assume that the lack of links between the trout and salmon in-dustries represents a substantial barrier to disease transmission, andhence contributes greatly to risk reduction.

Stocked trout fisheries are not included in the network (Munroand Gregory, 2009) although these may use trout from source farmsthroughout the UK. Fisheries were not officially registered at thetime for which the network was constructed, so the exact identityof individual fisheries could not be verified. However, although fishare moved onto such fisheries, they are not moved off so the fisheriesdo not form links between nodes in the farmed fish movement net-works (i.e. they are dead-end nodes) and therefore do not link thesalmon and trout movement networks. Furthermore, movement reg-ulations to fisheries in catchments containing salmon farms are beingstrengthened and these fishery operators will not be allowed tosource fish from farms under movement restriction (Richards, 2011).

Fish movements appear to be directly associated with many of theobserved cases of the spread of BKD in both trout (Section 3.2) andsalmon (Section 3.4) outbreaks. The lack of spread from “dead-end”trout farms (Section 3.1) is associated with lack of fish movements.Fish movements may occur over 100s of km so this is a critical com-ponent of BKD epidemiology.

4.5. Other anthropogenic networks

The movement of contaminated equipment poses the risk ofimporting or moving pathogens. The well boat network was shownto play a key role in transmitting ISAV during the Scottish 1998/9 ep-idemic (Murray et al., 2002) and fish transport lorries have been iden-tified as a potential risk factor for spreading fish diseases whentransporting fish from an infected farm if disinfection fails. However,as no UK based lorries transport fish between farms on the continentthey are not a risk factor for import of disease (Peeler and Thrush,2009).

The Shetland outbreak (Section 3.3) was confined to company Zand spatially scattered in a pattern that indicated anthropogenicspread. It did not, however, appear related to farmed fish movementsand therefore represents a different anthropogenic network with wellboats possibly playing a role. Bus-stop deliveries might be particularlyassociated with the spread of infection. Within the marine trout in-dustry in Loch Etive, farms with shared facilities became infected, al-though here spatial clustering is also a possible partial explanation.These anthropogenic networks appear to be mostly regional orsmall scale in structure and so probably spread pathogens over 10sof kilometres at most.

4.6. Hydrodynamic transport

Horizontal transmission of R. salmoninarum occurs via contact withskin or eyes (Hoffman et al., 1984) or consumption of faecal material(Balfry et al., 1996), so hydrodynamic transmission is possible. HoweverR. salmoninarum decays rapidly in water, Austin and Rayment (1985)found a decay of approximately 4 orders of magnitude in 4 days inunsterilised river water (equivalent to 10% h−1), while Balfry et al.(1996) found 60% decay after 8 h in raw seawater giving an hourlydecay rate of 11% h−1. Decay rates in sterile or filtered water aremuch longer. This is a rapid rate of decay compared to pathogens suchas IPNV (Toranzo and Hetrick, 1982). Many pathogens decay moreslowly when bound to particles than when free in the water (Sinton,2005) so R. salmoninarum bound to particles such as faecal material

might survive longer. However, faeces have been calculated to have asettling velocity of 0.017–0.06 ms−1 and using this estimate Gowenand Bradbury (1987) calculated that faeces are likely to sink out within200 m from a typical salmon farm. Hydrodynamic distribution dependson the faecal material breaking up into fine slowly sinking particles.

Transport risks depend not only on the survival of R. salmoninarumin the water, but also the rate of shedding from infected fish and min-imum infectious dose. These have been investigated for Chinooksalmon (Oncorhynchus tshawytscha) (McKibben and Pascho, 1999)and a minimum exposure of 7×102 R. salmoninarumml−1 for 24 hhas been established, however data are lacking for trout and Atlanticsalmon. Shedding would likely be elevated during clinical disease, asis the case with ISAV (Gregory et al., 2009). If this is the case thentransmission would be far more likely from a farm with BKD thanfrom one with sub-clinical R. salmoninarum infection.

Renibacterium salmoninarum appears to spread via the water be-tween cages in a farm (Wallace et al., 2011) and possibly betweenvery closely located farms (Sections 3.3 and 3.5); however water-borne spread did not appear to occur in Shetland (Section 3.4), al-though the also very labile ISAV did spread via the water betweensalmon farms separated by a few km in Shetland (Murray et al.,2010). The risk of spread of R. salmoninarum via this route cannotbe ruled out, but is likely to be limited to very short distances (100sof metres or a few kilometres) or special conditions that allow thetransport of faecal material.

4.7. Wild and escaped fish

Historically in Scotland BKD has been a disease of wild Atlanticsalmon (Mackie et al., 1933; Smith, 1964) however R. salmoninarumhas not been detected in wild fish since 1960 except in close associa-tion with infected Atlantic salmon (a single herring) and rainbowtrout farms (Wallace et al., 2011).

The estimated prevalence of R. salmoninarum in Scottish wild andescaped fish is extremely low (0.22%) (Wallace et al., 2011). Exten-sive background (not farm associated) surveillance, carried out by re-searchers from the Marine Laboratory, of 4520 wild freshwatersalmonid fish, from 1989 to 2004 returned no R. salmoninarum posi-tive results. In 2005–7 1143 wild fish were caught from waters dis-tant to aquaculture (so are not farm associated) and the screeningresults were negative. Over the same time period 1292 wild fishwere caught adjacent to infected farms, giving positive pools of 3-spined-stickleback (Gasterosteus aculeatus) (×2) and minnow(Phoxinus phoxinus) (×1) using qPCR (Wallace et al., 2011). Duringthis survey 268 escaped rainbow trout were also screened (232 ad-jacent to and 36 distant from aquaculture) giving three R. salmoni-narum positive qPCR pools from the adjacent location. However, asthe origin of these fish is not clear these results are difficult to inter-pret, other than to illustrate a possible R. salmoninarum transmissionmechanism. Other researchers report a possible association with R.salmoninarum infection between wild fish and rainbow trout farmsfrom English and Welsh rivers (Chambers et al., 2008).

Wild Arctic char were reported as high prevalence R. salmoninarumreservoirs in Iceland (Jonsdottir et al., 1998). Local populations of Arcticchar are found in many Scottish fresh water lochs and may form largeshoals. One such population is in Loch Arkaig and salmon smoltssourced from a farm (FS1) located in these waters showed a strong sta-tistical association with outbreaks in marine farms. However, the R. sal-moninarum infection status of Arctic char in Scottish lochs is unknown.

Inmarinewaters there is evidence for thepresence ofR. salmoninarumfrom ELISA screening of Pacific hake (Merluccius productus) (Kent et al.,1998) within a Chinook salmon farm and herring (unpublished data)from within a Scottish salmon farm undergoing a BKD outbreak(Section 3.3).

Wild fish may spend considerable amounts of time at one farm be-fore moving to another, possibly over distances of several km (Uglem

11A.G. Murray et al. / Aquaculture 324–325 (2012) 1–13

et al., 2009) and escaped fish may behave in the same manner. Thiscould enhance the risk of transport of pathogens between farms.

The use of hatcheries to enhance wild fish stocks may magnify theprevalence of any low-level R. salmoninarum infection (Fenichel et al.,2009). This would be of particular concern in situations where salmo-nid fish reared from wild parents were held in cages adjacent tofarmed salmonids. In such cases there is a risk of transmission if infec-tion were present in either population.

The very low reported prevalence of R. salmoninarum in Scottishwild fish suggests they are not an important reservoir or vector; how-ever, they have historically been important. Escaped rainbow troutmay pose a slightly higher risk and currently the status of Arcticchar is unknown.

5. Synthesis and conclusions

The risks associated with the different routes of R. salmoninarumspread are summarised using the information provided from the his-torical outbreaks and the analysis of contact structures within theaquaculture industry.

The hazard of interest is BKD in marine salmon farms, howeversince the introduction of R. salmoninarum to freshwater salmonfarms will be likely to spread to marine farms when fish are moved(probably to multiple farms) the hazard for our purposes is the intro-duction of R. salmoninarum to salmon farms.

We have identified that R. salmoninarum on salmon farms mightcome from activities within the salmon industry, from exposure towild reservoirs (although currently these appear small) or exposureto infection in the trout industry (Table 4).

We have established that the majority of cases can be traced backto movements within an industry or even within a company, mostlymovements of fish, in support of the conclusion of Austin and Ray-ment (1985). In the trout hatchery outbreak (Section 3.2) three ofthe four confirmed or suspect cases can be traced to movementsfrom infected farms. In the west coast outbreaks within company Y(Section 3.3) there are strong associations with movements of smoltsfrom particular freshwater farms. Therefore the farmed fish move-ment network (Green et al., 2009; Munro and Gregory, 2009) is crit-ical for the spread of R. salmoninarum.

Patterns of infection within Shetland suggest anthropogenicmovements related to industry activity but not to fish movements(Section 3.4). This might be in association with well boats visiting aprocessing plant (a similar route drove the spread of an outbreak ofISA in 1998/9 (Murray et al., 2002)) or the practice of ‘bus stop’ deliv-eries to the plant.

Table 4Empirically derived risk associated with different forms of transmission from observed Sco

Transmissionroute

Association of routewith existing BKDcases

Movement offarmed fish

Occurred repeatedly

Well boats Likely occurred inShetland

Movement ofwater or wild fish

Possible in several cases,certain between cages

Wild and escapedfish reservoirs

Arctic char possiblereservoir

Imported ova Potential risk, noevidence fromexisting cases

Imported parr and smolts Potential risk, noevidence from existingcases (EU only)

Transmission of R. salmoninarum through the environment almostcertainly occurs between individual cages in loch based trout farms. Itis possible that it could have occurred between neighbouring marinesalmon farms on the west coast but did not happen in Shetland asthere was no spatial clustering. The bacterium does not survive forlong periods in an unprotected (unbound) state (Austin and Ray-ment, 1985), so transmission requires attachment to non-sinkingparticles.

Wild and/or escaped fish might act as vectors for the transfer of in-fection from trout farms to salmon farms or between salmon farms. Afew R. salmoninarum qPCR positive escaped rainbow trout have beenfound around infected trout farms and while no wild/escaped posi-tive salmon have been found in Scotland since 1960 (Smith, 1964) aculture positive wild salmon has been found in England (Chamberset al., 2008). There is no conclusive evidence that transmission has oc-curred by this route, but it is a possible risk. Wild fish might also act asinfection reservoirs, this being a possible explanation for the failure offallowing at a trout farm (Wallace et al., 2011); however it is morelikely that this farm was re-infected from stocked farmed trout. Cur-rently there is no evidence for the presence of R. salmoninarum innon-farm associated Scottish wild fish and no cases of BKD in wildfish have been reported for decades.

Due to the transmission of infection between cages in loch basedfarms, and probably between ponds in land-based farms, eradicationof R. salmoninarum, as a minimum, should be based on fallowing atthe farm level (Wallace et al., 2011). The exception is a highly biose-cure tank based farm which has been cleared on a tank-by-tank basis.Generally R. salmoninarum is cleared from marine salmon farmswhich undergo fallowing at the farm-level between production cy-cles. However, even farm level fallowing has not been effective withtrout (Wallace et al., 2011), likely via the reintroduction with theinput of fish or from wild and escaped reservoirs.

The farmed fish movement networks for salmon and trout are al-most entirely separate. There is a class of mixed species farms, prob-ably in connection with sea-based trout farming, however theseappear mostly part of the salmon network and quite separate fromthe pure trout farm network.

There are a few areas in Scotland where salmon and trout farmsshare drainage basins however only five salmon farms are within5 km of a trout farm in the same catchment. There is no conclusive ev-idence for the transmission of infection between farms, but there isalmost certainly within-farm transmission (Wallace et al., 2011)and escaped fish (and to a lesser extent wild fish) may carry infection.Marine farms in adjacent areas of Loch Linnhe may have been ex-posed to R. salmoninarum from trout farms in Loch Etive.

ttish BKD outbreaks.

Trout to salmontransmission risk

Salmon to salmontransmission risk

Few risk contacts Most cases insalmon explainedby this route

Very unlikely dueto lack of contact

Likely associationwith some cases

Possible but notproven

Possible but notproven

Escaped infectedrainbow troutpossible vector

Possible, noevidence fromScotland,

Indirect effects Potential risk

Indirect effects Potential risk

12 A.G. Murray et al. / Aquaculture 324–325 (2012) 1–13

Put and take trout fisheries do not increase numbers of links in thefarmed fish movement network, but fisheries close to farms mightput these farms (whether trout or salmon) at risk of infection. Infor-mation on the location of fisheries is now being collected which willallow their distribution to be assessed. Recent controls have been in-troduced on movement of fish into put and take fisheries that sharecatchments with salmon farms (Richards, 2011).

If the R. salmoninarum prevalence in trout farms were to substan-tially increase this could increase the risk of transmission from thesefarms and thereby increase risk to salmon. Prevalence could be al-tered by imports or by changes in internal biosecurity practices andnetwork structure of the trout industry. As we have discussed thetrout and salmon industries do appear to exhibit a fairly high degreeof separation, however if the prevalence on trout farms was to changeradically then it is possible that these trout farms could become a rel-atively more important source of R. salmoninarum potentially causingsubstantially more outbreaks in salmon farms. The same applies if thegeographic structure of the trout industry were to change, perhapswith more trout farming in northwest Scotland.

If the prevalence of R. salmoninarum in salmon were to decrease tothe point of eradication then re-infection from trout, wild fish or im-ports could prevent final eradication or make any achievement tem-porary even if most infection is currently due to internal processeswithin the salmon industry. This may have already occurred as BKDhas disappeared from farmed salmon on occasions (although subclin-ical infection may well have persisted).

The increasing use of foreign imported salmon ova may increasethe risk of introductions of this vertically transmitted pathogen andhence decrease the relative risk from farmed trout. Imports of troutova might increase prevalence of R. salmoninarum in the trout indus-try thereby increasing risk from trout; this would only be significant ifimports substantially increased prevalence in trout. There is no evi-dence supporting a role for vertical transmission in the epidemiologyof BKD in Scotland, but the pathogen is truly vertically transmittedand with the lifting of AGs it is possible the risk from imports viathis route will increase.

The detection of R. salmoninarum in populations that are notexpressing clinical BKD may be problematic. Recent results suggestthe sensitivity of existing diagnostic surveillance testing may bepoor in cases of sub-clinical infection (Hall et al., 2011). Undetectedsources of infection may explain re-infection of fallowed trout farmsand infection of marine salmon farms on the Scottish west coast.

Alternative sampling regimes, perhaps using qPCR as the primaryscreening test (Hall et al., 2011), could improve detection of sub-clinical R. salmoninarum. However, if R. salmoninarum is widespreadin the network the consequence of such a rigorous policy could becostly, especially in the short term, as previously unknown cases ofsub-clinical infection may be detected. These costs might result inlimited benefits if these latent cases were in farms without off-sitemovements, especially in trout farms, but could be more usefulunder a risk-based approach targeted at farms with many off-sitemovements. A policy of only screening farms with clinical signs ofBKD, such as mortality or morbidity and listed diagnostic symptoms(Richards, 2011), and using qPCR as the diagnostic test has beenadopted. It is believed that this will achieve similar detection powerto the previous active surveillance regime using ELISA (Hall et al.,2011) while reducing diagnostic and sampling costs.

All the costs and benefits of controls need to be considered whenmaking decisions about BKD policy. The costs to industry of an eradica-tion programme can include culling and fallowing at infected farms. Theeradication policy increased the financial risks associated with runninga trout hatchery and thus decreases investment in this sector of the in-dustry; similar costs could apply to salmon hatcheries and smolt pro-ducers if surveillance were improved, as would probably be requiredfor eradication. Under compartmentalisation (with controls varied ei-ther between species or within geographically defined areas i.e.

compartments) BKD controlsmay bemore effectively targeted. Howev-er, compartmentalisation itself depends on implementation of strongmovement controls and surveillance within the pathogen-free zone.

Acknowledgements

Data for this paper was obtained fromMarine Scotland Fish HealthInspectors and scientists, from industry sources and the Scottish Envi-ronmental Protection Agency (SEPA). It was financially supported bythe Scottish Government through the project ROAME FC11105 and incollaboration with the Centre for Environment, Fisheries and Aqua-culture Science (Cefas) in review of UK BKD control policy.

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