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SID 5 Research Project Final Report

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NoteIn line with the Freedom of Information Act 2000, Defra aims to place the results of its completed research projects in the public domain wherever possible. The SID 5 (Research Project Final Report) is designed to capture the information on the results and outputs of Defra-funded research in a format that is easily publishable through the Defra website. A SID 5 must be completed for all projects.

A SID 5A form must be completed where a project is paid on a monthly basis or against quarterly invoices. No SID 5A is required where payments are made at milestone points. When a SID 5A is required, no SID 5 form will be accepted without the accompanying SID 5A.

This form is in Word format and the boxes may be expanded or reduced, as appropriate.

ACCESS TO INFORMATIONThe information collected on this form will be stored electronically and may be sent to any part of Defra, or to individual researchers or organisations outside Defra for the purposes of reviewing the project. Defra may also disclose the information to any outside organisation acting as an agent authorised by Defra to process final research reports on its behalf. Defra intends to publish this form on its website, unless there are strong reasons not to, which fully comply with exemptions under the Environmental Information Regulations or the Freedom of Information Act 2000.Defra may be required to release information, including personal data and commercial information, on request under the Environmental Information Regulations or the Freedom of Information Act 2000. However, Defra will not permit any unwarranted breach of confidentiality or act in contravention of its obligations under the Data Protection Act 1998. Defra or its appointed agents may use the name, address or other details on your form to contact you in connection with occasional customer research aimed at improving the processes through which Defra works with its contractors.

Project identification

1. Defra Project code F1150

2. Project title

The epidemiology of endemic and emerging diseases in freshwater fish populations in England and Wales

3. Contractororganisation(s)

CEFASBarrack RdWeymouthDT4 8UB          

54. Total Defra project costs £ 439565

5. Project: start date................ 02 January 2001

end date................. 31 March 2005

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6. It is Defra’s intention to publish this form. Please confirm your agreement to do so...................................................................................YES NO (a) When preparing SID 5s contractors should bear in mind that Defra intends that they be made public. They

should be written in a clear and concise manner and represent a full account of the research project which someone not closely associated with the project can follow.Defra recognises that in a small minority of cases there may be information, such as intellectual property or commercially confidential data, used in or generated by the research project, which should not be disclosed. In these cases, such information should be detailed in a separate annex (not to be published) so that the SID 5 can be placed in the public domain. Where it is impossible to complete the Final Report without including references to any sensitive or confidential data, the information should be included and section (b) completed. NB: only in exceptional circumstances will Defra expect contractors to give a "No" answer.In all cases, reasons for withholding information must be fully in line with exemptions under the Environmental Information Regulations or the Freedom of Information Act 2000.

(b) If you have answered NO, please explain why the Final report should not be released into public domain

Executive Summary7. The executive summary must not exceed 2 sides in total of A4 and should be understandable to the

intelligent non-scientist. It should cover the main objectives, methods and findings of the research, together with any other significant events and options for new work.The project began in mid February 2001 with the recruitment of an epidemiologist, Dr Edmund Peeler. The main task during the first year was to develop a four year research programme. The main drivers for the establishment of an epidemiology project was to introduce risk analysis methodology to the assessment of exotic disease threats and investigations to underpin the development of contingency plans for the control of exotic notifiable disease outbreaks. Work on assessing the potential of existing data on the occurrence of fish diseases and live fish movements (the Live Fish Movement Database - LMFD) also began. It was clear from the outset that the most important exotic disease threat to wild stocks in the UK was the monogenean, ectoparasite, Gyrodactylus salaris, and this became the focus of the import risk analysis work. Under the current regulations and practices, the main threat of introduction was from the movement of live fish transporters working in the UK and mainland Europe. The diseases risks from on-farm processing of imported fish carcasses were also highlighted. Further work on these risks is ongoing under a different contract (FB001). The work on G. salaris continued from the IRA study to investigating the spread of the parasite between river catchments. It was clear from the results of the study that live fish movements were the most important route of transmission. Other low risk routes of spread may spread the parasite to neighbouring rivers, and would be important to considered when developing contingency plans. The importance of live fish movements was quantitatively assessed in a stochastic model. Data from the LMFD was used. The outputs from the model provided estimates for the number of catchments likely to be affected in a G. salaris outbreak with time to first detection. This information can be used to support contingency planning, in particular resource requirements, for a G. salaris outbreak. The LMFD only stored data on the sites where fish had been moved, the frequency of movements and their dates were not recorded. Partly as a result of this work, the FHI now collect and store more detailed information on live fish movements.Surveillance has been an important element of this project. Current surveillance for disease in wild fish was reviewed and recommendations made to improve the detection of new and emerging diseases. The existing fish disease data was assessed to establish the frequency and geographic distribution of key diseases. It became clear that collaboration with the Environment Agency was necessary to improve our understanding of the endemic disease situation in wild fish populations. Discussions were initiated to store their disease outbreaks investigations in a database that would allow for analysis; however, a lack of EA resources prevented progress. In addition to working with the Fish Health Inspectorate on data analysis, Dr Sophie St-Hilaire (another epidemiologist working at CEFAS) worked with the FHI in the investigation of disease outbreaks, notably Vibrio anguillarum infections in Atlantic salmon in the Tyne and strawberry disease in farmed trout. At an international level the project established a system for monitoring electronic sources of new and emerging fish disease outbreak data. The primary function of this activity is to ensure early detection of disease events of significance to the UK.. Bulletins are regularly circulated within

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CEFAS, Defra and to outside bodies. The information is stored in a database, which is an increasingly valuable resource for the study of processes underlying disease emergence and spread.

A number of field studies were initiated in collaboration with other groups at CEFAS. An investigation of proliferative kidney disease (PKD) in south-west England was undertaken with the assistance of the EA. The aim of the study was simple: to investigate the within and between river prevalence of PKD. This information is fundamental to the design and analysis of future PKD studies in wild fish and may have potential to guide sampling regimes for other fish diseases. The results of this study are not yet available but will be completed under a new Defra contract. A similar study of a myxozoan parasite (Myxobolus buckei) of chubb was also initiated, again with the EA, to investigate the variation over time of the parasite within a river. This study was designed to assist in assessing the impact of infection on survival. Again, the results of this study are not yet available but will be completed under a new Defra contract.

In the latter part of the project the emphasis shifted to koi herpes virus (KHV). KHV is a potentially important emerging disease in the UK. The project aimed to address a number of important issues. Firstly, an ELISA test was developed to detect serum antibodies and thus provide evidence of past exposure using a non-destructive assay procedure. Secondly, experiments demonstrated that KHV can become latent and thus recovered clinically healthy fish are a potential source of infection. Infection in latently infected fish can be reactivated, virus is shed and naïve co-habited fish can become infected at temperatures above 200C. Polymerase chain reaction (PCR) and viral culture were not always able to detect virus in exposed fish or even clinically affected fish. These tools are unlikely to be effective at detecting a latent KHV infection. The development of the ELISA provided the necessary tool for a serological survey of farmed carp. Mapping the geographic distribution of the virus in England and Wales is fundamental to assessing the potential threat of the virus. To date 30% of the samples have been analysed, and no evidence of KHV has been detected. The antibody test can also be used to provide evidence of freedom from the virus in a cultured population, without the need for destruction of any stock; and therefore can contribute to the safe trade in live carp.

F1150 established epidemiology as a discipline within CEFAS. The project succeeded in working successfully with the Fish Health Inspectorate and research groups within CEFAS, and groups, such as the Environment Agency, outside of CEFAS. The project has needed to be flexible. Objectives have changed based on initial findings, the changing disease situation and Defra’s policy needs. The project has striven to make best use of the available data, improve the storage and collection of disease data and implement epidemiological studies of disease. The latter objective has also resulted in development and validation of diagnostic tests. Some of the projects objectives have not been met in full, in part because the F1150 was terminated 12 months ahead of schedule so that a replacement project would fall in line with the start date of other Defra funded projects.

Project Report to Defra8. As a guide this report should be no longer than 20 sides of A4. This report is to provide Defra with

details of the outputs of the research project for internal purposes; to meet the terms of the contract; and to allow Defra to publish details of the outputs to meet Environmental Information Regulation or Freedom of Information obligations. This short report to Defra does not preclude contractors from also seeking to publish a full, formal scientific report/paper in an appropriate scientific or other journal/publication. Indeed, Defra actively encourages such publications as part of the contract terms. The report to Defra should include: the scientific objectives as set out in the contract; the extent to which the objectives set out in the contract have been met; details of methods used and the results obtained, including statistical analysis (if appropriate); a discussion of the results and their reliability; the main implications of the findings; possible future work; and any action resulting from the research (e.g. IP, Knowledge Transfer).

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Assess routes of introduction of Gyrodactylus salaris to the UKIntroduction

Gyrodactylus salaris is a viviparous, monogenean freshwater parasite of salmon that naturally infects Baltic stocks of Atlantic salmon (Salmo salar) without causing clinical disease. However, in Atlantic stocks G. salaris is a serious parasite of pre-smolts. It multiplies unchecked by an immune response and death normally results (Bakke et al., 1990). G. salaris was introduced into Norway, probably via salmon parr imported from Sweden in the early 1970’s (Mo, 1994), and has resulted in the collapse of wild salmon populations in 44 Norwegian rivers (Mo, T.A, pers. comm.).

The impact of G. salaris in Norway had highlighted the importance of this parasite as a threat to wild Atlantic salmon stocks in the UK. G. salaris was the main focus of the Import Risk Analysis (IRA) work, which resulted in a peer-reviewed publication (Peeler & Thrush, 2004) and a number of posters and presentations. The current fish health regime prevents the importation of live salmonids from territories that have not substantiated freedom from G. salaris, and the IRA attempted to rank the routes of introduction given the current regulations (i.e. it was a restricted IRA).

Material and methods

The five stage approach to import risk analysis recommended by the Organisation International des Epizooties (OIE) was used (O.I.E., 2004);

i) hazard identification, ii) release assessment (description of pathways necessary for introduction), iii) exposure assessment (description of pathways necessary for the exposure of aquatic species in the importing country to the introduced exotic pathogen), iv) consequence assessment (identification of the consequences of disease introduction and establishment), and v) risk management (policies to reduce likelihood of introduction and mitigate the consequences).

The hazard is the introduction of Gyrodactylus salaris to the UK. The release and exposure pathways are considered for each route of introduction. The geographical distribution of the parasite and its biophysical properties were used to identify and assess potential routes of spread. The pathways of introduction fall into three main categories: importation of live fish and gametes, importation of eviscerated fish carcasses and mechanical transmission. The scenario tree for the introduction of G. salaris via rainbow trout carcasses is illustrated in Figure 1.

Results

The main findings were that the importation of other non-salmonids such as eels (Anguilla anguilla) represents a low risk because the likelihood of infection is very low and the parasite can survive on these hosts for no longer than 50 days. Importation of salmon carcasses presents a negligible risk because harvested fish originate from seawater sites and the parasite cannot survive full strength salinity. The importation of rainbow trout (Oncorhynchus mykiss) carcasses from G. salaris infected freshwater sites is likely to introduce the parasite, but establishment is only likely if carcasses are processed on a salmonid farm in the UK. A number or routes of mechanical transmission were considered (e.g. angling equipment, canoes, ballast water) and the most important was judged to be the movement of live fish transporters from farms on mainland Europe direct to UK fish farms.

Implications and future work

The opportunities for control and eradication of Gyrodactylus salaris are limited and the potential consequences are devastating. It is, therefore, crucial that the potential pathways for introduction are analysed to ensure that the appropriate policies to minimise the risk of introduction are in place.

Further work on the introduction of G. salaris (and other exotic pathogens) via the movement of live fish transporters and the imports of fish carcasses for processing on fish farms was continued under the contract FB001. In the future, territories may substantiate freedom from G. salaris and economic drivers for live salmonid imports may strengthen. Legal or illegal live salmonid imports would become the most significant risk of introduction. Since this work was completed changes in EU legislation allowed the importation of live salmon from seawater sites, given various caveats. The risk of G. salaris introduction via importation of live smolts to the UK was investigated under FB001 and a EU FP6 funded project (PANDA).

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Figure 1. Introduction of Gyrodactylus salaris via the importation of rainbow trout carcasses

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farm of origin Gs positive

prevalence (%) and abundance1 in harvested fish

prevalence (%) and abundance1 in processed carcasses

probability of survival during transport (%)

household use

solid waste liquid waste bait

processing/packing on farm

liquid waste solid waste

domestic sewage

land fill

F A R M E D A Q U A T I C E N V I R O N M E N T

direct discharge

leaching/scavenging/seepagedirect discharge

W I L D A Q U A T I C E N V I R O N M E N T

picnics

possible pathways

unusual pathways

low significance pathways Gs = Gyrodactylus salaris1 number of parasites per infected fish

leaching/scavenging

further processing/packing

release assessment

exposure assessment

Assess the routes of dissemination of G. salaris between river catchments in England and WalesIntroduction

A natural continuation of the G. salaris IRA work was to investigate routes of transmission of the parasite between river catchments in the UK (Peeler et al., 2004). If the parasite were introduced into the UK, the focus of a control programme will be to minimise the risk of spread of the parasite to uninfected rivers (especially, those with wild salmon populations). Experience in Norway indicates that G. salaris spreads throughout an entire river catchment within 2 years of the parasite first being detected on juvenile Atlantic salmon (Johnsen & Jensen, 1991). It has been suggested that the infection is spread upstream by migrating adult salmon which infect resident juveniles (Soleng et al., 1998).

We used risk analysis (Anon., 1993) to identify and rank all possible routes of transmission of G. salaris from infected to uninfected rivers in England and Wales, to support contingency planning for an outbreak of G. salaris and to identify areas for further research.

Materials and methods

The identified hazard is the spread of parasite G. salaris once introduced into England and Wales. The OIE recommended approach to IRA was adopted (i.e. 5 stage IRA, for details see above). Biophysical properties of the parasite (section 4) initially were used to identify potential routes of spread. The ranking of the routes used several different information sources, including experience of G. salaris spread in Norway and live-fish movements in the UK. Data on live-fish movements and fish production for 2001 (collected by the Fish Health Inspectorate (FHI, CEFAS) were used. The risk of exposure was considered for each of the routes of introduction. For each route the number of catchments are risk and the frequency of events leading to release or exposure were estimated. An assessment of the likelihood release, exposure and combined release and exposure for each pathways was made.

Results

The results of both the release and exposure assessments and the number of catchments at risk have been used to estimate and rank the overall importance of each route (Table 1).

Table 1. Assessment of the importance of routes of transmission for Gyrodactylus salaris between river catchments in England and Wales

Route of G. salaris transmission between river catchments (unit)

Release assessment

Exposure assessment

Combined assessment

probability event results in

introductionc

probability introduction

results in establishment

importance rank

live rainbow trout or salmon very high very high extremely high 1

other species of live-fish - non-salmonid fish, grayling and brown trout

high high high 2

movement of salmon between rivers

moderate high low 3

farm equipment, staff and vehicles

low low low 4

effluent from a fish processing plants

high low low 5

angling equipment low low very low 6

canoes / boats etc. low low very low 7

rainbow trout / salmon eggs (disinfected)

low low very low 9

eel migration low low-to-moderate extremely low 10

piscivorous birds extremely low extremely low negligible 11

Movement of rainbow trout and salmon represents the most-important route of transmission for a number of reasons. Firstly, movement of rainbow trout or salmon from an infected site is highly likely to lead to transmission. Secondly, there are large-scale movements of rainbow trout between farms and from farms into fisheries across England and Wales (thus the parasite could achieve a widespread geographic distribution quickly). The

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movement of other species of fish is also very likely to lead to transmission (however, prevalence and abundance of infection is lower than for rainbow trout or salmon). All the other routes will result primarily in local spread to neighbouring rivers, and spread will be less rapid (although they might become important over many years).

Discussion

Bakke et al. (1992) proposed four transmission routes: 1) direct contact between living infected and uninfected fish, 2) contact between living and dead infected fish, 3) contact between fish and detached parasites on a substrate, and 4) contact between uninfected fish and detached parasites in the water column. The main routes of transmission from an infected to an uninfected catchment are through the movement of live infected fish and mechanical transmission of free-living parasites on fomites (inanimate objects or substances capable of transmitting infectious organisms).

Within months of the introduction of G. salaris the movement of live-fish, particularly rainbow trout, is highly likely to result in the wide geographic dissemination of the parasite in England and Wales. The movement of species other than Atlantic salmon or rainbow trout is only a significant risk if they originate from sites holding Atlantic salmon or rainbow trout. Over a period of many years mechanical transmission (mainly via boats, angling equipment and the movement of vehicles and people between farms) and migrating salmon will spread the parasite from an infected river to neighbouring rivers.

If the parasite is not detected rapidly after introduction into the country, the movement of rainbow trout could lead to widespread dissemination and thus a multi-focal outbreak. Control of the UK Foot and Mouth Disease (FMD) outbreak in 2001 was difficult because the virus had been widely disseminated before the infection was detected and bans on livestock movements imposed. One of the main reasons why FMD was not detected rapidly was that the Pan-Asia O strain caused few lesions in sheep (Davies, 2002). This is analogous to G. salaris infections in rainbow trout: generally subclinical and unlikely to be detected by farmers.

Recommendations and future work

Contingency plans to minimise the spread of the parasite must prioritise, firstly, the regulation of live-fish movements and, secondly, measures to prevent local spread. Contingency planning for FMD had not considered a multi-focal outbreak scenario (Anderson, 2002). It is vital that this lesson is learnt and G. salaris contingency plans are based on multi-catchment scenarios.

It was clear that the movement of live-fish was potentially the most important route of dissemination. We have concluded that future, quantitative work should focus on stochastic modelling of live fish movements. This work will provide assessments of the likely extent of an outbreak and geographic distribution of the parasite with time to first detection. The number of salmon rivers infected largely will determine the consequences of an outbreak.

Other ad hoc risk analysesAn Evaluation of the Relative Risks of Infectious Salmon Anaemia Transmission Associated with Different Salmon Harvesting Methods in Scotland

A collaboration with scientists at the Fisheries Research Services (FRS), Aberdeen on the assessment of the risks of spreading infectious salmon anaemia (ISA) by different harvesting methods resulted in a peer-review publication (Munro et al, 2003). Risk analysis methodology was used to rank different harvesting methods. The key findings were that the most serious risks of ISA transmission was associated with holding live fish in cages, known as harvest stations, adjacent to processing plants and the discharge of untreated effluent from processing plants. It is recommended that holding live fish in harvest stations near the processing plant is phased out. Effluent from processing plants should be treated to inactivate ISAV and other pathogens. This measure has been effective in reducing the number of ISA outbreaks in Norway. Well-boats carrying live fish may disseminate pathogens, including ISA virus, if they exchange in the vicinity of fish farms and the recommended disinfection and ballast discharge procedures are not followed. Towing cages of live fish to a harvesting station at a processing plant is the harvesting method most likely to spread ISA virus. Cages are only likely to be towed to a harvest station if the distance between the farm and the harvest station is relatively short. A quantitative approach is needed to provide a more accurate assessment of the risks associated with harvesting. The results of this qualitative assessment are adequate for the development of recommendations to reduce the spread of ISA virus at harvesting.

Transmission of Renibacterium salmoninarum - - an analysis of routes of spread to farmed rainbow trout (Oncorhychus mykiss) and from farms to wild fish populations in England and Wales – a report for the Committee for Aquaculture Health May 2002.

Both qualitative and quantitative approaches have been adopted to assess the increased risk of spread of Renibacterium salmoninarum (Rs) to rainbow trout farms, and from farmed rainbow trout to wild fish populations. Rs is the causative agent of bacterial kidney disease (BKD). The disease was first reported as causing disease in Atlantic salmon (Salmo salar) on the River Dee, Scotland in the 1930’s (Smith, 1964). The first case in farmed fish occurred in rainbow trout (RBT) (Oncorhychus mykiss) in Scotland in 1976 (Sanders & Fryer, 1980). The disease has been reported in Europe, Japan and North America. BKD is a systemic disease, with a preference for kidney tissue, which occurs in both the marine and freshwater life stages of anadromous salmonids. Rs has been found

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inside the ovum, and vertical (Elliott et al., 1995) and horizontal (Balfry et al., 1996) transmission have been demonstrated. It is generally considered that, unlike many fish pathogens, Rs is an obligate fish pathogen, and not an opportunistic infection. In the UK BKD is a notifiable disease and is a List III disease under EU legislation (Council Directive 91/67/EEC). The Department of the Environment, Food and Rural Affairs (Defra) operates a control policy on the belief that the disease is not widespread in Great Britain yet poses a significant threat to both farmed and wild fish populations. A risk analysis approach was adopted to assess the likely increased spread of Rs between RBT farms, and from farms to wild fish, if the disease was deregulated. The relative importance of the different routes of spread of Rs are considered.

A Monte Carlo simulation using the @RISK software (Palisade Corp. Newfield, NY, US) has been adopted to estimate the maximum output of Rs that is likely to be found in the outflow from an infected rainbow trout farm. The model was run 1000 times and latin hypercube sampling was used. The highest, lowest and most likely estimates of input parameters were modelled as pert distributions (Vose, 2001). Sensitivity regression analysis was undertaken to identify the most important input parameters. The main routes of Rs introduction to rainbow trout farms were identified : i) purchase of live fish, ii) purchase of eggs, iii) inflow of water and iv) direct contact with infected wild fish or escaped rainbow trout. The purchase of live infected fish or infected eggs are probably the most important routes of introduction. The mean concentration of Rs in the outflow from an infected rainbow trout farm was 3.7 x 10-8 cfu / litre (SD = 3.1 x 10-8). The top 5% of outputs were greater than 1.0 x 10-7 cfu / litre (the highest estimate was 3.2 x 10-7 cfu /litre). The distribution of Rs concentration was a good fit to a gamma distribution. The output was most sensitive to the following inputs in a regression sensitivity: percentage of infected fish (0.70), concentration of Rs in the faeces of infected fish (0.41), and waterflow per tonne of trout (-0.35). Whilst transmission via oral intubation of 2.5 x 105 cfu of Rs has been demonstrated in chinook salmon, data are not available to estimate a minimum infective dose.

The main risk of Rs introduction into a rainbow trout farm, after the introduction of infected stock or eggs, probably originates from infected wild fish or their carcasses entering the farm. However, there is no evidence that this is a common problem. The main risk to wild fish is the introduction of infected hatchery reared fish. It is not possible to reach any definitive conclusions regarding the importance of rainbow trout, farmed for table production, as source of infection for wild fish. Since the Rs outflow from an infected farm is low, it poses little threat to farms downstream and the escape of fish from a farm is probably a more important source of infection to wild populations.

Review surveillance for disease in wild freshwater fish populations in England and WalesA review of the current disease surveillance in wild freshwater fish populations was undertaken. A number of recommendations to improve surveillance were made. The main focus of the current fish disease surveillance programme in England and Wales is the maintenance of the approved zone status for exotic notifiable diseases. Only very limited sampling of wild fish is required and, therefore, little is known about the disease status of these fish stocks. To better understand the role of disease in wild fish populations would require changes to the current system. Several suggestions ranging from educational programmes for anglers, creation and maintenance of a database that utilizes data collected from Defra-funded research projects, identifying catchments that are at higher risk of having notifiable or new diseases and including these in the current surveillance programmes, investigating the cause(s) of declining fish populations, and using sentinel fish farms on catchments to monitor endemic pathogens could be made to improve the current system. Deciding which strategy to take should be based on the objectives of the surveillance programme and the funding available. Furthermore, evaluation of any changes to the current system should be conducted for their efficacy, and any shortcomings should be addressed.

Assess and summarize the existing disease data for freshwater fish in England and Wales : making recommendations for improvement where necessary. Introduction

Historic monitoring and surveillance data contained on the CEFAS Live Fish Movement Database (LFMD) was used to assess the known distribution of economically important freshwater fish diseases. The objective of this work was to identify areas that are free of disease and areas of unknown disease status (i.e. not tested) in addition to mapping disease positive sites.

Methods

Customised structured query language (SQL) reports were constructed to interrogate the LFMD for reported proliferative kidney disease (PKD), BKD, koi herpes virus (KHV) and spring viraemia of carp (SVC) pathology. Results were exported to Excel spreadsheets and the data cleaned using a Visual Basic macro. Disease distributions were visualised using GIS software (MapInfo) including river boundary catchment data, wild salmonid distribution and fish farm site data as additional layers.

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Figure 2. The distribution of economically important freshwater fish diseases in England and Wales

(Grey lines are river catchment boundaries)

Results & Discussion

The distribution of the diseases was mapped (Figure 2). These data identify where the diseases occurred but cannot be used to establish disease free areas (systematic sampling based surveillance is required).

The current reporting methods used for recording information on the LFMD do not allow effective surveillance work as attempted here. Information returned by SQL interrogation can only be used after extensive and time-consuming data cleaning. The main constraint was use of free text fields to record recognised pathologies. Data retrieval methods frequently relied on the presence of key words, which may be used to record either the presence or absence of a particular condition. This means that each record returned has to be checked manually to establish the context in which the key word has been used. Although this may be acceptable for tens or hundreds of records, it is not an adequate procedure of the assessment of many thousands of results.

A recommendation was made that diagnoses for specific conditions should recorded in a boolean format on the LFMD, and chosen from drop down menus to facilitate future searches and remove the possibility of misinterpretation of pathologists comments.

Future work: Environment Agency database (LFMD-Extension)

Fresh water wild fish disease surveillance in England and Wales is primarily conducted by CEFAS and the Environment Agency. The Agency conduct investigations into the causes of wild fish mortalities and also undertake targeted sampling of fish for Category II parasite and any gross signs of disease prior to live fish

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movements into mandatory waters in England and Wales (these are health assessments required Section-30 of the Salmon and Freshwater Fisheries Act, 1975). The objective of their sampling programme is to protect wild fisheries from pathogens considered a threat if introduced to a naïve population of fish. The results of both the disease investigations and the health checks provide a resource of information on pathogens affecting wild fish.

At present CEFAS and the EA maintain separate disease databases with differing degrees of complexity. The systems cannot be easily amalgamated and data can only be summarised separately. It would be advantageous to both agencies to analyse and summarise the data using a co-ordinated approach. CEFAS uses the LFMD for all relevant data relating to fish farm registration, fish imports and exports, fish movements and the keeping of non-native fish in England and Wales and, through the Fish Health Database, as a laboratory information management system. The EA use the LFMD for fish removals and Section 30 introductions, however, no laboratory test results are recorded in the system. Information on LFMD relating to fish mortalities is limited to results of diagnostic tests performed at CEFAS (usually virology only). Data on all of the other diagnostic tests undertaken by the National Fisheries Laboratory (NFL) is stored in a separate Environment Agency database or as paper records.

From an epidemiological point of view database amalgamation would allow Improved data storage, analysis, and monitoring and provide an opportunity to examine trends in pathogen or disease distribution. This would also allow the sharing of data with academic organisations and the development of collaborative projects focused on the analysis of CEFAS and NFL data. The recording and sharing of fish health information will facilitate inter-agency co-operation in the monitoring of diseases and parasites found in England and Wales. This will improve disease surveillance efforts and help identify areas and pathogens that require more research, control strategies or monitoring.

Progress

The EA agreed in principle to the benefits that extended functionality of the LFMD would provide, and discussions were initiated to establish how this could be progressed. The necessary LFMD modifications required (in both data capture and storage) for incorporating mortality investigations and routine health checks were reviewed in detail. The estimated cost was submitted to the EA, but insufficient funds were available in FY2004-05. Discussions are ongoing.

As an interim solution, a MS-Access database was developed by CEFAS to log the results of outbreak investigations undertaken by the EA. Although the database was relatively simple in design, such an approach would give CEFAS staff access to information collected by the EA and allow a basic epidemiological assessment to improve understanding of the distribution of some fish pathogens across England and Wales. To date one year’s data (61 outbreaks in 2000) have been transcribed from paper records by EA staff, who have agreed to provide more data in the future.

Summary of disease outbreaks investigated under FC1150 in 2003 / 2004.One of the objectives of F1150 was to strengthen the epidemiological aspect to disease investigations of new and emerging diseases. All disease outbreak investigations were conducted in conjunction with the Fish Health Inspectorate and the Environmental Agency.

1. Ulcer Disease associated with Aeromonas salmonicida in two carp fisheries. Findings will be published in Fish Veterinary Journal (submitted Jan. 2004). Findings have also been presented at the Fish Veterinary Society meeting in Dec. 2003 Ulcer disease, a bacterial infection of the skin, also referred to as erythrodermatitis was diagnosed in two independent carp fisheries in the UK during the summer of 2003. The managers from these two fisheries reported over 30% mortality in their fish. Affected fish included mirror carp (Cyprinus carpio) and a hybrid carp, which was thought to be a cross between crucian carp (Carassius carassius) and common carp. Clinical signs associated with this disease include varying sizes of skin ulcers, exophthalmia and ascites . In severe cases, scales protrude to give the fish a pinecone appearance, often referred to as dropsy. Although there are usually several types of bacteria isolated from the skin ulcers of affected fish, certain strains of Aeromonas salmonicida are believed to play the most significant role in the disease (Elliot & Shotts, 1980). These bacteria are classified as atypical subspecies of A. salmonicida (Hoole et al., 2001), and in carp they are generally not found in organs other than the skin (Wiklund & Dalsgaard, 1998). Mortality in affected fish is thought to occur because of osmoregulatory failure (Noga, 2000). Transmission trials conducted at CEFAS, where affected fish from one of the fisheries with ulcer disease were transported back to the laboratory and cohabited with naïve carp, suggested that other factors besides the presence of the bacteria may play a role in the severity of this disease. In our trial only 2 of the 40 naïve fish developed small ulcers, and none of the fish had ascites or died. In fact, some of the original affected fish recovered once they were in our facility. At the end of the trial, atypical A. salmonicida was confirmed on the skin of 4 of 12 original fish, and in 1 of the 2 naïve fish with ulcers. Although it was not possible to confirm why the severity of the disease was less in a laboratory setting than the natural environment several factors such as water quality, fish density, different feed rations, lack of natural predators, and lack of water temperature fluctuations may have

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played a role. In both outbreaks reported to CEFAS in 2003, there was the possibility of transport stress prior to the disease occurring. Fisheries managers should minimize the level of stress on the their fish as much as possible.

2. Vibrio outbreak in Atlantic salmon in the river Tyne. Findings will be published in the Fish Veterinary Journal (submitted Jan. 2004). The cause of the fish mortality on the River Tyne in the summer of 2003 was attributed to Vibrio anguillarum. Infection with this pathogen was most likely exacerbated by the low water flow, which impaired fish movement upstream and lead to large aggregations of fish, and high water temperatures, which increased the replication rate of the bacteria and stressed the fish. Both of these environmental conditions would have favoured fish to fish transmission of the bacteria and increased the mortality rate associated with the bacteria. Vibrio sp have been detected in fish from this river on other occasions when pre-spawning mortality was higher than expected and this may be a recurring problem. Given the trend in climate change, revision of the water and fisheries management strategies may be warranted to reduce the effects of this bacteria in this system. Answers to critical questions such as: whether sea trout are also dying of the bacterial infection; where the fish are initially infected with the bacteria; and the migration behaviour of the Atlantic salmon and sea trout in the river, would assist fisheries mangers in controlling losses from this disease. In the interim, some considerations should be given to management practices that reduce the amount of secondary spread (fish-to-fish) of the bacteria, such as the aggregation of fish and the amount of contaminated carcasses in the river. With an early warning system in place it would be possible to predict whether this bacteria might result in elevated mortality. Management plans can then be altered based on the assessed risk of a disease outbreak.

3. Strawberry disease in farmed rainbow trout. Findings from our investigation have lead to the hypothesis that this condition may be associated with Aeromonas hydrophila skin infections. Findings have been presented at the Fish Veterinary Society meeting in Dec. 2003 and in a short article in Trout News. The recent preliminary examination of fish in the UK with the condition did not find systemic infections with bacteria; however, several bacteria types were isolated from the skin lesions. One of these, which was consistent in all affected fish cultured was Aeromonas hydrophila. These bacteria are commonly found in the aquatic environment and usually are considered non-pathogenic; however, under certain conditions they can produce haemolytic toxins (Howard et al., 1996). The histological findings reported for strawberry disease are not inconsistent with the tissue reaction that would be observed with a haemolytic toxin (i.e. inflammatory cell response with no evidence of a pathogen and areas of haemorrhage). During our investigation of strawberry disease in the summer of 2003, it was observed that the raceways with the highest prevalence of the condition were also those on re-used water. A similar observation has been made previously (Kfoury et al., 1996a; Kfoury et al., 1996b). These authors speculated that re-used water contained a higher bacterial load and ammonia concentration, and lower pH and oxygen, all of which could promote disease in fish. It may be possible that these environmental parameters are adequate to promote A. hydrophila toxin production or activation, if the bacteria have the appropriate gene(s). Unpublished data that supports this hypothesis includes the fact that in 1999 affected fish transferred to our research facility with good water quality were not able to transmit the infection and, in fact, recovered from the lesions relatively quickly after transfer (L. Richards, personal communication CEFAS). A proposal to test the hypothesis and determine control measures for the condition was submitted to the British Trout Association and to the French Farmers Association. Isolates have been sent to the University of Reading for further analysis under their A. hydrophila research programme.

Design and maintain a database containing monthly updates of worldwide emerging fish and shellfish diseases.Introduction

A monitoring programme and database for emerging fish and shellfish diseases reported around the world was established. The objectives are to track global trends and, more specifically, to identify and provide advanced warning of, disease threats that may affect wild and farmed fish stocks in the UK. A systematic approach has been adopted to search a range of “grey” or “soft” literature sources, including Internet newsletters, alerting services and news agency releases. These sources provide information on a more real-time basis compared with peer-reviewed publications. In addition, letters and short communications to journals as well as information from personal research and industrial contacts have been included

An emerging disease has been defined as a new disease, a new presentation of an old disease (e.g. increased severity or appearance in a new species) or an existing disease that appears in a new geographical area. Over the past 20 years a significant number of new diseases have emerged in human and animal populations, and their study has become an increasingly important area of research in both human and veterinary medicine. New and emerging diseases have caused substantial economic and environmental impact and the importance of emerging diseases in the aquatic environment and in has been recognised. For example the emergence of infectious salmon anaemia (ISA) cost the Scottish farming industry £20-million in the 1998/9 outbreak

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Material and methods

Reports of new or emerging diseases were identified by a routine daily surveillance of the electronic sources listed in Table 2. Additional records were collected from peer-reviewed journals, trade journals and personal communications from research, government and industrial sectors. Only one data source for each entry, identifying the first report of an emerging disease event detected by the surveillance programme, was recorded on the database. The information was stored in a Microsoft Access-97 database (Microsoft Corporation, Seattle, WA). Data is exported to a Microsoft Excel-2000 spreadsheet for analysis.

Results and discussion

In the first year of the surveillance, a total of 62 emerging disease developments, involving 43 different pathogens or unidentified causes, were identified in 22 countries. Events involving viruses and parasites accounted for the large majority of data collected (48% and 24% of the total respectively). Eight reports of bacterial disease and one fungal infection were also logged. Known diseases occurring in new locations were found to be the most important emerging disease category, accounting for 60% of the data; and 20 out of 41 of these new locations were the result of non-contiguous spread. Eight completely novel diseases were reported, and two known diseases were found in new species. The aetiologies of five fish-kill events were unidentified.

Reports of emerging disease were dominated by events in salmonid species (45%). Results were evenly split between marine and freshwater environments and 65% of all records were associated with farmed populations. Most of the results were obtained from Europe and North America (82% collectively). There were no reports of emerging diseases from some geographical regions with significant aquaculture production.

Table 2. Sources of electronic information and surveillance actions

Source Address

Office International des Epizooties (OIE) www.oie.int

FAS Program for Monitoring Emerging Diseases (ProMED)

www.promedmail.org

IntraFish (Media service) www.intrafish.com

FIS (Media service) www.fis.com

Scientific forum on fish and fisheries

(Fish-Sci)

segate.sunet.se/archives/fish-sci.html

University of Guelph Plant & Agriculture surveillance archives: (AnimalNet, FS-Net)

www.plant.uoguelph.ca

Centre for Emerging Issues (CEI) www.aphis.usda.gov

National Wildlife Health Centre www.nwhc.usgs.gov

ProMED provided the most information on emerging diseases (37%), and the majority of these reports originated from North America. News agencies and information services, including IntraFish and FIS, accounted for a further 27% of the results and provided more information on European disease events. Only 32 out of the 62 reports provided either the date or month of disease outbreak, so a robust assessment of outbreak time was not possible and no clear month-by-month trend was evident.

No reports of emerging or new diseases were posted from some regions with significant aquaculture production, possibly a result of under-reporting. This regional bias is also reflected in the species analysis; most reports are from salmonid species which are cultured in large-scale, intensive production systems in developed economies (mainly in Northern Europe, Canada and Chile) where disease monitoring and reporting is likely to be more efficient compared with countries producing non-salmonids (for example carp) in small-scale pond culture.

Information collected by this monitoring programme is providing a useful first stage of screening for emerging fish diseases that may be potential threats to the UK. The accumulation of long-term data will also allow a more rapid and authoritative assessment of the significance of new and emerging diseases, especially their likely future impact. Emerging diseases present a problem to regulators. In general, legislation only exists for the statutory control of known notifiable diseases. It is therefore important that systems exist to monitor and investigate the occurrence of emerging diseases and, when deemed necessary, make notifiable and control. Additionally, the database that has been generated provides a useful resource for the investigation of the processes underlying

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disease emergence in fish. The value of the database will increase with time, and in the future will provide a resource for investigating long-term trends in disease emergence.

Future work

This work will continue under a new Defra funded project. Access to the database will be extended through development of a web-based version.

Develop a model that identifies catchments at risk of disease transmission based on probability of fish movements into those catchments.Introduction

The protection of farmed and wild salmonids depends on effective contingency plans for the elimination of exotic diseases should an outbreak occur. The movement of live animals is frequently the most important route of spread, especially long distance spread, for an introduced disease. A qualitative risk analysis has identified the anthropogenic movement of live Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss) as the most important route for the transmission of G. salaris between river catchments (Peeler et al., 2004) indicating that quantitative data is urgently required. To address this, the movement relationships between the farm sites registered for salmonid production (rainbow trout, brown trout and Atlantic salmon) in England and Wales were established and stochastic modelling techniques were used to provide input to an ongoing epidemiological assessment of the likely spread of introduced pathogens through the movement of live fish.

Material and methods

The Live Fish Movement Database (LFMD) was interrogated by standard structured query language (SQL) reports to determine all destinations of live fish movement in 2002 from each of the 261 farms registered on the database in April 2004 for holding stocks of salmonid fish. The results were exported to MS-Excel spreadsheets, transformed into standardised arrays and combined to provide a movement relationship matrix.

A stochastic model was developed to project a single site-to-site contact scenario over a 52-week period within a MS-Excel spreadsheet. For the purpose of the simulation, binomial distribution functions were used to determine when each site made a fish movement (for example, the sale of fish to another site) and destinations for movements were predicted using site-unique discrete-probability distribution equations generated directly from the movement relationship matrix.

Simulations were achieved using @Risk (Palisade Corporation, Newfield, NY), a risk analysis software add-on for MS-Excel. The sampling method was Latin hypercube and a total of 522,000 iterations were run using individual exporting sites (index sites) seeded with a positive status (generating 2610 scenarios for each of 200 exporting sites). Positive status was passed to naïve sites by forward movement contact from the index site through successive weeks of the scenario. Additional contacts were generated by movements from any farm subsequently acquiring positive status, either directly from the index site, or via one or more intermediaries. This process was automated by a Visual Basic control macro, which substituted a different farm as the index site at the start of each new iteration. On completion of each iteration, a separate macro was used to appended the contact status of all farm sites, non-farm destinations and their corresponding river catchments at 3, 6, 9 and 12 months to output files.

Results

Live fish were transported from 200 farm sites registered for the production of salmonid fish to a total of 1653 freshwater destinations (farm and non-farm) in England and Wales in 2002. The distribution of river catchments contacted during simulation 12 months are shown in Figure 3. The median number of catchments contacted after 3 and 12 months were 16 and 53, respectively. In 5% of simulations 63 or more catchments were contacted, and in 1% of simulations 75 or more catchments were contacted after 12 months. The worst-case scenarios, predicted by the model, were 31 and 104 catchments contacted after 3 and 12 months respectively.

A framework of outbreak categories have been overlaid on the catchment contact distributions in Figure 1. Degrees of severity were assigned to outbreak scenarios as low, moderate and severe for the involvement of up to 3, 4-10 and 11 or more catchments respectively. Assuming an introduced disease remains undetected for 3-months the risk of a severe outbreak (spreading to more than 10 river catchments) was 7%. However, if the disease was to go unnoticed for a year, this risk increased to nearly 90%.

River catchments were ranked in order of the likelihood of the sites within them receiving consignments of live fish from other farm sites. The Severn, Trent and Thames river catchments were identified by simulation to be the most likely to receive consignments of live fish.

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Figure 3. Distribution of river catchments contacted after 12 months from single index farm inputs

Discussion

This is the first attempt to quantitatively assess the likely spread of an exotic fish pathogen within England and Wales via the movement of live salmonid fish. Time to diagnosis of an exotic or emerging disease in the UK will be a critical factor in determining its geographical spread before control and eradication efforts can be initiated. The results clearly demonstrate that, unless a disease is detected and diagnosed quickly, and measures to prevent spread implemented, a multi-focal outbreak involving many river catchments is highly likely

This work will inform the contingency planning process for the introduction and control of exotic aquatic pathogens in England and Wales. Effective contingency plans must be based on realistic scenarios and importantly include a worst-case scenario. The results indicate that contingency plans for G. salaris control in England and Wales should be based on multi-focal outbreaks and wide geographical spread on first detection. The UK FMD epidemic in 2001 highlighted the need for planning to include a worst-case scenario.

Results from the model have allowed us to rank catchments by their likelihood of receiving a potentially infected consignment of live fish during an outbreak. The live fish movement model described here provides outputs that form a sound basis on which to base contingency planning and risk-based surveillance for exotic notifiable fish diseases. It is a an important first step in the modelling of aquatic disease spread on a national basis and provides a vital component of a future, more comprehensive disease transmission model.

Future work

Modelling the spread of exotic fish diseases will continue under a Defra funded post-doctoral project based at the University of Liverpool (FC1153).

Determine the within- and between-river variation in prevalence of brown trout infected with Tetracapsuloides bryosalmonaeIntroduction

This project was undertaken with Defra funded project F1138. Proliferative kidney disease (PKD), a disease of salmonids caused by the myxozoan parasite Tetracapsuloides byrosalmonae, has been the subject of considerable research at CEFAS. It affects both farmed (Feist, 1997) and wild fish (Feist et al., 2002). Studies of this disease in wild fish requires knowledge of the within and between river variation in prevalence. These data are crucial to calculating sample sizes and interpreting results.

Material and methods

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0

5

10

15

1 7 13 19 25 31 37 43 49 55 61 67 73 79 85 91 97 103

Number of catchments contacted

Per c

ent o

f tot

al it

erat

ions

Low level outbreak = 7.5%

Moderate outbreak = 3.8%

Severe outbreak = 88.7%

Worst-case senario = 104 catchments contacted

Probability of worst-case senario = 0.000192%

Approximately 320 brown trout were sampled from 16 sites on 6 rivers in South West England. These are currently being evaluated for T. bryosalmonae. This work was undertaken in collaboration with the Environment Agency.

Results

Histological slides have been processed, however, the reading of slides has not yet been completed. This work will continue under the new Defra funded project (F1168).

Determine the within-and between river variation in prevalence of chub infected with Myxobolus buckei over timeItroduction

In recent years a myxosporean parasite M. buckei has been observed in the vertebral column of fry in several species of coarse fish (work undertaken in Defra project F1138). Chub fry in several rivers in Yorkshire appear particularly affected (Feist, pers. comm). Pathological changes associated with this parasite consist of spinal cord damage and reduction in vertebral disc space. It is unlikely that fish recover from these changes and, is it therefore hypothesized that infection with this parasite could affect the survival of fish during their first winter.

Objective:

It is possible that M. buckei infection of chub (Leucicus cephalus) is associated with elevated first winter mortality. If this were the case we would expect to see a similar prevalence of parasite or signs of pathology associated with the parasite in the 0+ fish sampled in the fall and the same year class of fish sampled one (or two) years later. Also, the strength (or success) of a year class should be independent of that year class’s infection status with M. buckei. The objective of this study of )+ fish was to measure the within and between river variation in prevalence to assist in the design of future investigations (e.g. sample size calculation) and aid in the interpretation of existing data.

Material and methods

This research was a collaborative project with CEFAS (F1138 and F1150 projects) and the EA. Five to seven rivers in the EA’s coarse fish fry monitoring programme in the county of Yorkshire with a history of M. buckei in chub fry, and five to seven rivers in the same area without a history of the parasite were selected. In collaboration with the EA’s coarse fish monitoring programme, 0+ chub were collected, measured, necropsied, body condition scored, and histologically examined for M. buckei from each of the rivers. The prevalence and intensity of infection with M. buckei in 0+ chub in 2003 will be determined from the fish collected during this study.

Results

The histological slides have not yet been all been examined. This work will be completed under a new Defra funded project (F1168)

Koi herpese virus investigations: latency, transmission, antibody production and sero-surveillanceObjectives:

a) determine whether a carrier state exists for Koi Herpsevirus (KHV)

b) validation of serological tools for KHV

c) conduct a serological survey for exposure to KHV in coarse fish farms and fisheries in England and Wales

Introduction

Koi herspesvirus (KHV) is a newly recognized virus associated with mortality in common carp (Cyprinus carpio carpio) and koi carp (Cyprinus carpio koi) in the United States, Israel, South Africa, the European Union and Japan and South-East Asia. This virus has been reported in England, but it is not considered endemic. With the number of koi carp imports to England and the high number of fish movements within the country, there is concern that this virus will be introduced to common carp fisheries and become widespread. The impact of KHV on naïve wild common carp populations in the UK is unknown, but the virus has severely affected wild populations in other countries (e.g. Japan and Thailand).

It is unlikely that clinically diseased fish would be transferred into a fishery. However, fish that have previously been exposed to the virus and recovered may be moved to fisheries. If KHV infection can result in a subclinical latent carrier state, survivors of an outbreak can potentially shed virus at a later date. An important characteristic of herpesviruses is their ability to establish latency in their natural hosts, including those with natural or vaccine induced immunity. Latent virus remains dormant and non-infectious for long periods, but can be reactivated to become pathogenic with the host subsequently showing clinical signs, and on occasions mortality. The mechanism for reactivation of herpesviruses remains unknown; however, it is believed that the host’s physiological state plays an important role. The reactivation of virus may result in horizontal transmission and a new outbreak.

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Little is known about latency with KHV. If KHV is similar to other herpesviruses, such as cyprinid herpesvirus (CHV), and channel catfish virus (CCV), then a latent state is likely to occur. Survivors of a disease outbreak with these two other viruses become asymptomatic carriers with specific neutralising antibodies and can excrete virus. A practice that has been used in the Koi carp industry to induce immunity to KHV is to expose fish to the virus at non-permissive temperatures (less than 17 C and greater than 28 C). Data suggest that at the high end of the non-permissive temperature range, exposed fish develop specific immunity and upon re-challenge, are protected. However, there is much debate as to whether this strategy of inducing a 'natural immunity' produces latent infections in fish and whether these fish can subsequently transmit the virus to naïve fish.

The objectives of this study were, firstly, to determine if a latent state of KHV exists in common carp and whether the virus can be transmitted to naive fish by cohabitation. The second objective was to investigate the use of specific antibody production in carp as a means to demarcate exposed populations of fish by Enzyme Linked Immunosorbent Assay (ELISA) in both laboratory challenge experiments and a serological survey of farms and fisheries in the UK to assess the spread of the disease (wild fish). This work was carried out in collaboration with Defra funded project F1136.

Methods

To investigate latency and re-activation of KHV infections, two populations of carp were exposed to the virus and subsequently co-habited with naïve fish. The first population was exposed to KHV and the population was split into a low and a high temperature scenario. The high temperature scenario fish were kept at the exposure temperature of 21C and subsequently co-habited with a population of naïve fish after mortality had subsided. Control fish followed the same regime. In the low temperature scenario carp were exposed at 21C and quickly lowered to 11C, a temperature not permissive for pathogenesis. After six months at this temperature, a population of naïve fish was added and the tank and the temperature was raised to 23 C. Control fish followed the same regime.

The second population of fish were exposed to KHV at 21C and held at that temperature. After a month, the temperature was gradually lowered to 11C and the fish were split into two groups. Naïve fish were added to only one tank at this temperature after 6 months and the temperature was subsequently raised, in both tanks, to 23 C. Control fish followed the same regime.

After exposure, a sample of fish from each exposure tank and all control tanks was taken monthly and analysed by ELISA for specific antibody to KHV. The KHV ELISA was based on an ELISA developed for channel catfish virus (Crawford, 1999). Considerable work was required to make the test suitable for KHV antibody (KHVabEIA). The development utilised blood samples taken during the KHV-exposure studies. Serum samples from carp exposed to KHV gave high absorbance values in the ELISA at dilutions of 1:3200 and above. The criteria used for a fish to be considered positive for KHV antibodies was an absorbance reading at the 1:1600 dilution that was greater than the average for the negative control plus three times its standard deviation, and if all other lower dilutions had higher absorbance readings than the previous dilution. Using these criteria antibodies were observed in the fish from the experimental tanks after the initial disease outbreak had subsided. The antibody response was then used to monitor for exposure to KHV when cell culture and PCR techniques were not reliable at detecting virus in sub-clinical fish. Carp were sampled monthly for 9 months after the re-emergence of the virus and antibodies were detected at each sampling with a prevalence of between 20 and 40%. All control fish tested before exposure to KHV were negative for KHV-specific antibodies by ELISA and none of the control fish tested during the study (n=10) and at the end of the study (n=10) had antibodies to KHV.

To assess the distribution of kio herpes virus in England and Wales, thirty-fish samples were purchased from farms with as wide a geographic range as possible. All farm sites registered on the LFMD for stocking common or mirror carp (Cyprinus carpio) were contacted by telephone and asked to supply fish for the survey. Returns were therefore limited to the availability of suitable fish (20-30g fish that had been resident on the farm for a minimum of 6 months were required) and the consent of the farm owner (farms are not obliged to supply fish for non-statutory sampling). Live fish samples were transported to the Weymouth Laboratory by courier where possible. Of the 153 farms contacted, 21 provided samples to test for previous exposure to the virus. In addition, seven 30-fish samples were provided by the Environment Agency collected from fish submitted from fisheries for Section-30 health checks.

Results

The findings in this study suggest that common carp exposed to KHV can, under some circumstances, become latently infected with the virus. Furthermore, they can shed the virus and infect naïve fish if co-habited for a period of time at temperatures above 200C. Given the findings of this study the practice of exposing fish to live virus in an attempt to immunise the fish should be used with caution especially if these fish are to be transferred to other locations and co-habited with naïve fish.

KHV re-appeared in two out of four experimental exposure tanks. The initial mortality in these populations when exposed to the virus appeared to be a function of temperature. Temperature appeared to be a major factor in the re-activation of a covert or latent infection. Second outbreaks of KHV were only observed in tanks containing KHV survived fish that were above 200C, a permissive temperature for the pathogenesis of KHV.

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Apparent latent infections were not seen to be a function of initial mortality as second outbreaks were observed after high and low initial mortality. Also, given that not all exposed fish showed a re-emergence of disease, it is likely that only a small percentage of fish become carriers from an exposed population.

The virus re-emergence was several months after exposure and in one tank caused a mortality 54% in previously exposed fish and 100% mortality in naïve fish. In the second tank where re-emergence was observed, there were no naïve fish yet all the previously exposed fish died.

Enzyme Linked Immunosorbent Assay (ELISA) was a useful tool in identifying exposed populations of fish. Polymerase chain reaction (PCR) and viral culture were not always able to detect virus in exposed fish, or even in some cases clinically diseased fish. Using these tools, it is unlikely that a latent KHV infection would be found. Antibody to KHV was detected in experimental fish and was consistent with exposure. Seroconversion in common carp experimentally exposed to KHV was between 40 – 60% and was more rapid in the fish that were held for longer at a higher initial temperature. Antibodies were detected when there were no signs of disease and the response appeared to last at least fifteen months after exposure.

Analysis of the carp serum samples has been delayed mainly due to lack of reagents. Samples from 7 farms have been tested to date and all are negative. This objective is being carried forward under another Defra funded project (F1167).

Future work

KHV studies are continuing under a new Defra funded project (F1166)

Objectives due for completion in 2005/6 and carried over to the follow-up projects:Development of a method of analysing disease data from surveys to substantiate freedom from disease becomes and objective of F1165.

Assessment of the impact of climate change on the health status of fish in England and Wales becomes an objective of F1165.

ReferencesAnderson, I. 2002. Foot and Mouth 2001: Lessons to be learnt inquiry report (London, The Stationery Office), p.

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Anon., 1993, Risk analysis, animal health and trade. Rev Sci tech Off Int Epiz 12, 434.

Bakke, T.A., Harris, P.D., Jansen, P.A., Hansen, L.P., 1992, Host specificity and dispersal strategy in gyrodactylid monogeneans, with particular reference to Gyrodactylus salaris (Platyhelminthes, Monogenea). Dis Aquat Org 13, 63-74.

Bakke, T.A., Jansen, P.A., Hansen, L.P., 1990, Differences in the host resistance of Atlantic salmon, Salmo salar L., stocks to the monogenean Gyrodactylus salaris Malmberg, 1957. J Fish Biol 37, 577-587.

Balfry, S.K., Albright, L.J., Evelyn, T.P.T., 1996, Horizontal transfer of Renibacterium salmoninarum among farmed salmonids via the fecal-oral route. Dis Aquat Org 25, 63-69.

Davies, G., 2002, The foot and mouth disease (FMD) epidemic in the United Kingdom 2001. Comp Immunol Microbiol Infect Dis 25, 331-343.

Elliot, D.G., Shotts, E.B.J., 1980, Aetiology of an ulcerative disease in goldfish, Carassius auratus (L.): experimental induction of the disease. J Fish Dis 3, 145-151.

Elliott, D.G., Pascho, R.J., Palmisano, A.N., 1995, Brood stock segregation for the control of bacterial kidney disease can affect mortality of progeny chinook salmon (Oncorhynchus tshawytscha) in seawater. Aquaculture 132, 133-144.

Feist, S.W., 1997, Pathogenicity of renal myxosporeans of fish. Bulletin of the European Association of Fish Pathologists Weymouth. 17, 209-214.

Feist, S.W., Peeler, E.J., Gardiner, R., Smith, E., Longshaw, M., 2002, Proliferative kidney disease and renal myxosporidiosis in juvenile salmonids from rivers in England and Wales. Journal of Fish Diseases. 25, 451-458.

Hoole, D., Bucke, D., Burgess, E., Wellby, I., 2001, Diseases of Carp and other Cyprinid Fishes. Blackwell Science Ltd, Oxford.

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Howard, S.P., MacIntyre, S., Buckley, J.T., 1996, Toxins in the genus Aeromonas, In: Ed Austin, B., Altwegg, M., Gosling, P.J., and Joseph, S (Ed.) John Wiley & Sons, Ltd, pp. 267-277.

Johnsen, B.O., Jensen, A.J., 1991, The Gyrodactylus story in Norway. Aquaculture 98, 289-302.

Kfoury, J.R., Okamoto, N., Tanaka, M., Yoshimizu, N., LaPatra, S.E., Maita, M., 1996, Rash skin disease on rainbow trout. Fish Pathology, 31, 197-201.

Mo, T.A., 1994, Status of Gyrodactylus salaris problems and research in Norway, In: Lewis, J.W. (Ed.) Parasitic diseases of fish. Samara Publishing, Tresaith, Dyfed, Wales, pp. 43-48.

Noga, E.J., 2000, Fish Disease Diagnosis and Treatment. Iowa State University Press, Ames, Iowa.

O.I.E., 2004, Aquatic Animal Health Code, 7th Edition, Paris, 167 p.

Peeler, E.J., Gardiner, R., Thrush, M.A., 2004, Qualitative risk assessment of routes of transmission of the exotic fish parasite Gyrodactylus salaris between river catchments in England and Wales. Preventive Veterinary Medicine 64, 175-189.

Peeler, E.J., Thrush, M.A., 2004, Qualitative analysis of the risk of introducing Gyrodactylus salaris into the United Kingdom. Dis Aquat Org 103-113, 103-113.

Sanders, J.E., Fryer, J.L., 1980, Renibacterium salmoninarum gen. nov., sp. nov., the causative agent of bacterial kidney disease in salmonid fishes. Int J Syst Bacteriol 30, 496-502.

Smith, I.W., 1964, The occurrence and pathology of Dee disease. Freshwater and Salmon Fisheries Research 34, 1-12.

Soleng, A., Bakke, T.A., Hansen, L.P., 1998, Potential for dispersal of Gyrodactylus salaris (Platyhelminthes, Monogenea) by sea-running stages of the Atlantic salmon (Salmo salar): field and laboratory studies. Canadian Journal of Fisheries & Aquatic Sciences 55, 507-514.

Vose, D., 2001. Qualitative versus quantitative risk analysis and modelling. In: OIE International Conference on Risk analysis in Aquatic Animal Health, Paris, 8-10 February 2000, pp. 19-26.

Wiklund, T., Dalsgaard, I., 1998, Occurrence and significance of atypical Aeromonas salmonicida in non-salmonid and salmonid fish species: a review. Dis Aquat Org 32, 49-69.

References to published material9. This section should be used to record links (hypertext links where possible) or references to other

published material generated by, or relating to this project.

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Peer-reviewed publications

Munro, P.D., Murray, A.G., Fraser, D.I., Peeler, E.J., 2003, An evaluation of the relative risks of infectious salmon anaemia transmission associated with different salmon harvesting methods. Ocean Coast Manage 46, 157-174.

Peeler, E.J., in press, The role of risk analysis and epidemiology in the development of biosecurity in aquaculture. Diseases in Asian Aquaculture.

Peeler, E.J., Gardiner, R., Thrush, M.A., 2004, Qualitative risk assessment of routes of transmission of the exotic fish parasite Gyrodactylus salaris between river catchments in England and Wales. Preventive Veterinary Medicine 64, 175-189.

Peeler, E.J., Thrush, M.A., 2004, Qualitative analysis of the risk of introducing Gyrodactylus salaris into the United Kingdom. Dis Aquat Org 103-113, 103-113.

Thrush, M.A., Peeler, E.J., submitted for publication-a, Monitoring the emergence of fish and shellfish diseases using electronic sources. Dis Aquat Org.

Thrush, M.A., Peeler, E.J., submitted for publication-b, Stochastic simulation of live fish movement in England and Wales to predict potential spread of exotic pathogens. Dis Aquat Org.

S. St-Hilaire, N.Beevers, K.Way, R-M. Le Deuff, P. Martin and C. Joiner. Reactivation of latent infections of KHV in common carp Cyprinus carpio (submitted for publication, Dis Aquat Org)

St-Hilaire, S. Ulcer Disease associated with Aeromonas salmonicida in two Carp fisheries (accepted by the Fish Veterinary Journal, Jan. 2004)

St-Hilaire, S Vibrio outbreak in Atlantic salmon in the river Tyne. (accepted by the Fish Veterinary Journal Jan. 2004).

St-Hilaire, S. and Jeffery, K. (2004) Stawberry disease in farmed rainbow trout, Trout News 37, p24. http://www.cefas.co.uk/publications/troutnews/tnews37.pdf

Other reports

Infectious Fish Disease Epidemiology: its potential to improve fish health management in the UK - a report for the Fisheries Science Customer Group. December 2002, Edmund Peeler and Ron Stagg

Transmission of Renibacterium salmoninarum - - an analysis of routes of spread to farmed rainbow trout (oncorhychus mykiss) and from farms to wild fish populations in England and Wales – a report for the Committee for Aquaculture Health May 2002. Edmund Peeler

Wild fresh water fish disease surveillance in England and Wales, 2002, Sophie St-Hilaire

Posters

Peeler, E.J. (2001) Quantitative import risk analysis – a desirable and practical goal for aquaculture? (presented at the European Association of Fish Pathologists, Dublin, September 7-13, 2001)

Peeler, E.J. (2002) A qualitative risk analysis for the introduction of Gyrodactulus salaris in the UK. (presented at the annual meeting of the Society for Veterinary Epidemiology and Preventive Medicine, Robinson College. Cambridge University, 3-5 April 2002).

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Peeler, E.J., Thrush, M.A. and Gardiner, R. (2003) A preliminary risk analysis for the transmission o fhte exotic fish pathogen Gyrodactylus salaris between river catchments in England and Wales. (presented at the 10th Sypmposium of the International Society for Veterinary Epidemiology and Economics, November 11-17, 2003, Vina del Mar, Chile).

Thrush M.A. and E.J. Peeler E.J. (2004) Monitoring emerging aquatic animal diseases. (presented at the Society for Veterinary Epidemiology and Preventive Medicine Annual Meeting March 2004

Presentations:

Edmund Peeler

The risk of the introduction and spread of Gyrodactylus salaris in England and Wales. 14 November 2002, Fish Veterinary Society Autumn Meeting, Edinburgh.

The role of risk analysis and epidemiology in the development of biosecurity for aquaculture (2002). Presented at the 5th meeting on Diseases in Asian Aquaculture, held at Surfers Paradise, Australia, 25-29 November 2002.

Emerging animal diseases – why are they important? Presented at a workshop on emerging disease held at the Veterinary Laboratories Agency, 30 January, 2003.

Sophie St-Hilaire

"Ulcer Disease associated with Aeromonas salmonicida in two Carp fisheries." Submitted to Fish Veterinary Journal Jan. 2004. Findings have also been presented at the Fish Veterinary Society meeting in Dec. 2003.

"Vibrio outbreak in Atlantic salmon in the river Tyne." Submitted to the Fish Veterinary Journal Jan. 2004.

"Stawberry disease in farmed rainbow trout," presented at the Fish Veterinary Society meeting in Dec. 2003 and in a short article in Trout News.

Mark Thrush

Using modelling techniques to predict the spread of disease by farmed live fish movement in England and Wales. 11th International Conference of the EAFP. Malta, September 2003.

A preliminary risk analysis for the transmission o fhte exotic fish pathogen Gyrodactylus salaris between river catchments in England and Wales. 11th International Conference of the EAFP. Malta, September 2003.

Modelling the movement of live salmonids in England and Wales. University Fish Disease Modelling Consortium Stakeholder meeting. Leahurst, Wirral. 23rd September 2004.

Stochastic simulation of live fish movement to predict the spread of exotic aquatic pathogens. Society for Veterinary Epidemiology and Preventive Medicine Annual Meeting March 2005.

ThesesMaster of Research Thesis: Koi herpesvirus in the common carp: studies on latency, transmission and

serology Mr N.D. Beevers – University of Plymouth

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