general enquiries on this form should be made...

General enquiries on this form should be made to:Defra, Science Directorate, Management Support and Finance Team,Telephone No. 020 7238 1612E-mail: [email protected]

SID 5 Research Project Final Report

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 FC1151

2. Project title

Risk to the aquaculture industry and the environment associated with current and emerging bacterial and fungal fish/shellfish diseases and their treatment

3. Contractororganisation(s)

Cefas Weymouth LaboratoryBarrack RdThe NotheWeymouthDorset

Postcode DT4 8UB

54. Total Defra project costs £ £278,970

5. Project: start date................ 14 June 2001

end date................. 31/03/06

SID 5 (2/05) of 27

6. It is Defra’s intention to publish this form. Please confirm your agreement to do so.....................................................................................YES X 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.

SID 5 (2/05) of 27

One of the important functions of Cefas is to investigate new and emerging diseases, including those caused by bacteria and fungi of farmed and wild fish in England and Wales on behalf of Defra. Project FC1151 enabled Cefas Weymouth Laboratory to both undertake multidisciplinary investigations into emerging microbial diseases as they were reported and also to maintain its general expertise in microbiology fish disease research in order that this competence is available to Defra in the event of an emergency.The project had five main objectives:

Objective 1.0 To maintain an excellent level of awareness of all emerging microbial pathogens of fish and shellfish that are of relevance to the UKAccess to comprehensive and current information is key to the evaluation and perception of new diseases, including those caused by bacteria and fungi. The Fish Health Inspectorate (FHI) report large scale fish kills in wild and farmed fish respectively. Cefas also has existing links with FRS Aberdeen, with whom details on emerging diseases were exchanged on a regular basis. This is formalised under the CFRD Aquatic Epidemiology Working Group. There was also a regular exchange of information with the Fish Veterinary Society, researchers from Stirling Institute of Aquaculture and other institutes working on emerging bacterial and fungal diseases, anglers and angler groups and the Environment Agency. The published literature (both through journals and websites) was scanned as part of existing procedures. All data were logged into the Emerging Disease Database that has been already set up under Defar contract F1150. Project staff also attended a number of international scientific meetings where experiences and data were shared with other researchers.Throughout the project, routine literature and internet searches were conducted to identify all recently recognised bacterial and fungal fish and shellfish pathogens that may be of relevance to the UK. Discussions were also held with fish veterinarians and fish health experts. This information was used to determine which diseases were investigated further under Objectives 2 and 3.

Objective 2.0 To develop diagnostic capabilities for emerging bacterial and fungal pathogens of fish and shellfishDiagnostic capabilities were developed for a range of microbial pathogens. These included: 02.1 Identification of Gram positive cocci pathogenic for fishMany Gram-positive cocci (GPC) have been described as being responsible for disease in a range of freshwater or marine fish species. In this project we compared different commercially supplied rapid identification systems, for their ability to identify accurately a panel of test bacteria that included 21 culture collection reference strains and two field isolates. Results indicated that Biolog GP was the best system for this purpose.Cefas Weymouth is now in a position to rapidly identify Gram positive cocci responsible for outbreaks of disease in wild and farmed fish populations.

02.2. Culture and Diagnosis of Piscirickettsia salmonis and Rickettsia-like organisms Techniques for growing and storing different strains of the pathogen responsible for Piscirickettsiosis, on a range of different cell lines, were successfully developed. We also successfully challenged Atlantic salmon with this disease and used infected material to develop an antibody based system for detecting the pathogen in samples of diseased salmon (IFAT). Cefas Weymouth laboratory is now in a position to rapidly diagnose outbreaks of disease caused by P. salmonis and has gained potentially valuable experience of working with Rickettsia-like organisms.

02.3. Identification of Candidatus arthromitus .Rainbow trout gastroenteritis (RTGE) is a serious condition which was increasingly reported from UK farms during the reporting period. As well as undertaking outbreak investigations, rainbow trout diagnosed with clinical signs were examined for the presence of C. arthromitus, the segmented filamentous bacteria (SFB) purportedly responsible for the condition. Although we have visualised the presence of SFB in the guts of infected fish, the molecular methods employed were not able to determine whether these were C. arthromitus or not.

02.4. Identification of emerging Yersinia ruckeri strains During the course of this project, increased incidences of the bacterial disease Enteric Red Mouth were diagnosed in farmed salmonids in the UK. Characterisation of responsible strains, coupled with outbreak investigations by Cefas staff, indicated that the disease was caused by emergent strains of Y. ruckeri to which fish, previously vaccinated against the disease, were no longer resistant to.

02.05 Development of 16S rRNA gene based identification techniques. FC1151 was used to establish and validate a 16S rRNA gene based identification protocol in the Weymouth Laboratory for both Gram negative and Gram positive bacteria.

SID 5 (2/05) of 27

Objective 03. To investigate and assess the impact of new or emerging bacterial and fungal pathogens of fish and shellfishA range of diseases were investigated during the course of the project using a multidisciplinary approach.

03.1.Susceptibility of selected freshwater course and salmonid fish species to Lactococcus garvieaeLactococcocis caused by the Gram positive bacterial pathogen L. garvieae is a significant threat to farmed rainbow trout. In a series of experiments we investigated the susceptibility of three salmonid species (Atlantic salmon, brown trout and grayling) and seven cyprinid fish species (carp, tench, rudd, barbel, chub, dace and roach) to L. garvieae. We showed that grayling were highly susceptible to L. garvieae (>40% mortality when exposed to infected rainbow trout) and many of the course fish species tested were at least partially susceptible. 57% of rainbow trout that survived a cohabitation challenge were still shown to be carrying L. garvieae up to 27 Days post-infection. There was also some evidence that other fish species may also be able to carry L. garvieae.

03.2. Investigations into the causes of a new severe chronic skin condition affecting Atlantic salmon ( Salmo salar l.) Pre-smolts There have been reports of severe losses post-vaccination by some salmon farmers with losses generally attributed to fungal infections. We also noted high incidences of mortality post-vaccination in particular stocks of salmon in trials. Results of disease investigations and challenge trials indicated that these mortalities might actually be linked to a bacterial pathogen (a Chryseobacterium sp.), rather then to fungal infection specifically.

03.3. Virulence of Candidatus arthromitus in rainbow troutA tank-based trial was undertaken in an attempt to determine whether RTGE can be transmitted from infected rainbow trout to naïve fish that have not previously been exposed to this disease. Two different transmission routes were tested with fish exposed to gut contents of RTGE infected fish and also by direct contact (cohabitation). The overall aim of the study was to better inform policymakers and farmers of the likely risks of the condition being transmitted. The outcome of the trial was inconclusive, with RTGE not being successfully transmitted, although there was a high mortality in the naïve fish attributed to another bacterial disease, furunculosis, caused by Aeromonas salmonicida subsp. salmonicida. RTGE remains a significant concern, so it is advised that further work be undertaken to better determine the disease’s aetiology with a view to developing effective control strategies.

03.4 Studies on Red Mark Syndrome (RMS)In 2005 there were outbreaks of a skin disease of farmed rainbow trout, known as Red Mark syndrome, in England and Wales. Epidemiological analysis indicated that the disease was infectious and had spread through live fish movements from Scotland to England. The affected fish presented with a range of lesions of differing severity. However, despite extensive analysis, including histopathological investigation of preserved material (by light and electron microscopy), as well as mycology, bacteriology and virology, no single potential disease agent could be consistently isolated from affected fish. Data from transmission trials showed that the disease could be transmitted from affected fish to naïve rainbow trout in the laboratory. Future work will depend on the incidence of this disease and therefore its risk to cultured and wild fish stocks.

Objective 4: Establishment of the Bacterial Culture Collection (BCC) and contribution to the Registry of Aquatic Pathology (RAP) DatabaseThe Weymouth Laboratory BCC is a valuable National archive of aquatic bacterial and fungal strains. 2969 isolates are now listed on the BCC, almost 200 of them being national collection isolates. 799 isolates in total have been added since the beginning of 2001.Amongst other material, samples from L. garvieae, C. arthromitus, S. galactiae, P. salmonis and Red Mark Syndrome infected fish have been added to the RAP.

Objective 5: Validation of antimicrobial susceptibility tests for all major fish pathogens Standardised antimicrobial test methods were validated against a range of relevant aquaculture pathogens and were, with the possible exception of L. garvieae, found to be acceptable. These methods are now used as part of routine diagnostic procedures in the Weymouth Laboratory.

Main ImplicationsThe main implications of this study are that diseases caused by bacterial and fungal agents remain a highly significant, possibly increasing, threat to farmed and wild fish populations in England and Wales. The project provided Defra with timely advice on the risks posed by a number of different diseases, including: Lactococcosis, rainbow trout gastroenteritis, Red Mark syndrome and a skin disease of salmon. The ability of the Weymouth laboratory to diagnose emerging diseases caused by bacteria and fungi was also improved (e.g. tools for the identification of Gram positive and rickettsia bacterial pathogens were validated). The laboratory now routinely uses molecular based identification methods as a consequence of this project.

SID 5 (2/05) of 27

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).

Objective 01.Objective as set out in the contract: ‘To maintain an excellent level of awareness of all emerging microbial pathogens of fish and shellfish that are of relevance to the UK’

Extent to which objective 1 has been met. This objective was fully met over the course of the project.

Details of the methods used and the results obtained, including statistical analysis (where appropriate) and a discussion of the results and their reliability.Access to comprehensive and current information is key to the evaluation and perception of new diseases, including those caused by bacteria and fungi. Cefas has existing links with FRS Aberdeen, with whom details on emerging diseases were exchanged on a regular basis. This is formalised under the CFRD Aquatic Epidemiology Working Group, although there is considerable informal exchange of information between FRS and Cefas Weymouth.. There was also a regular exchange of information with the Fish Veterinarians Society. Cefas staff have contact with researchers from Stirling Institute of Aquaculture and other institutes working on emerging bacterial and fungal diseases. Cefas has good links with anglers and angler groups, who represent an important source of information on wild fish and fisheries. The Environment Agency and the Fish Health Inspectorate (FHI) report large scale fish kills in wild and farmed fish respectively. The published literature (both through journals and websites) was scanned as part of existing procedures. All data was logged into the Emerging Disease Database that has been already set up under F1150.

Throughout the project, routine literature and internet search were conducted to identify all recently recognised bacterial and fungal fish and shellfish pathogens that may be of relevance to the UK. Discussions were also held with fish veterinarians and fish health experts. Identified threats included:

01.1 Streptococcosis Streptococcosis caused by various Gram positive cocci, including Streptococcus iniae, S.faecalis, S.difficilis, S.parauberis, Lactococcus garvieae, etc. has been a serious problem in Japanese aquaculture for at least twenty years and in Israel since the early 1980’s, but has only recently become a major threat worldwide. In Europe, the disease is reported to cause the trout industry major losses in Spain, Italy & France. Streptococcus iniae (formerly S.shiloi) has been reported as a cause of Streptococcosis in both freshwater and marine fish species in several Mediterranean countries, Canada, the USA, and Australia. L.garvieae (formerly E.seriolicida) is now the cause of the most serious economic losses among farmed Rainbow trout in Italy, with mortality up to 50% in market sized fish. The pathogen affects many fish species, both marine

SID 5 (2/05) of 27

and freshwater in Europe, Japan, the Mediterranean, Australia and the USA. Both S.iniae and L.garvieae can cause invasive disease in humans after skin injuries during the handling of fresh-farmed fish. There is also evidence that S. iniae can be transmitted from wild fish to cultured stocks ( Zlotkin,-A.; Hershko,-H.; Eldar,-A. 1998). Vagococcus salmoninarum has been reported as causing significant losses of salmonids in France Australia and North America, and Staphylococcus spp, Lactococcus piscium among others have been reported as pathogens of various fish species worldwide. We also reported Streptococcus agalactiae as the cause of the massive fish kill that affected the wild fish population in Kuwait Bay in summer 2001 (Cefas report, Algoet et al., 2001). It seems likely that pathogenic Gram positive cocci will continue to increase in importance with the diversification of fish species farmed in Europe and worldwide. In the UK, although most of these organisms are still exotic, trout are known to be susceptible to many Gram-positive cocci. The virulence of these pathogens in wild fish, such as UK coarse fish, was not known so work was undertaken under this project to assess this particular risk (see Objective 3.1 later).

01.2 Pathogenic Vibrio spp. Several new fish and shellfish pathogenic Vibrio species, including V.scopthalmi, V.natriegens, V.penaeicida, V.gazogenes, V.trachuri and V.viscosus (proposed for re-classification as Moritella viscosa) have been identified in recent years. Several species thought not to be fish pathogenic or previously found mainly in shellfish or Eel, such as V.vulnificus, V.damsela, V. alginolyticus, V. cholera, V. harveyi, V.pelagius, V splendidus, V.fluvialis and V.parahaemolyticus have recently been isolated from both freshwater and marine fish. It is likely that with improvements in taxonomy and in isolation/diagnostic techniques, many more Vibrio species will be implicated in diseases of farmed fish and shrimp. Work was done under this project to improve Cefas Weymouth Laboratory’s ability to diagnose diseases caused Vibrio spp. (Objective 02.5)

01.3 Atypical Aeromonas salmonicida Atypical A. salmonicida has been isolated from a large number of marine and freshwater fish species worldwide, and has recently been reported in wild-caught Wrasse used as cleaner fish in Scotland. Aeromonas hydrophila continues to be reported as the cause of systemic infections in catfish, rainbow trout, carp and ornamentals worldwide. Other Aeromonas spp. including A. sobria, A. caviae and A. jandaei have been associated with septicemias in both freshwater and marine fish species. With the growth in ornamental fish culture and the likely expansion (e.g. in Germany) of edible carp farming, A. hydrophila and others are likely to pose a significant threat in Europe and worldwide.

01.4 Rickettsias: Piscirickettsia salmonis has caused important losses (up to 80%) of salmonid fish in Chile since 1989, and Rickettsia-like organisms have been isolated from a wide range of fish and shellfish species worldwide. Work was initiated under this project to improve Cefas Weymouth Laboratory’s ability to diagnose rickettsiosis in farmed and wild fish (see Objective 02.02).

Other diseases with a likely or possible microbial aetiology associated with fish losses in the UK:01.5 Candidatus arthromitus C. arthromitus may be responsible for rainbow trout gastroenteritis (RTGE). This disease, associated with the presence of a non-cultivable segmented filamentous organism, was first described in 2001 (Urdaci et al., 2001) but was likely to have been present on farms in France and Spain for many years, leading to severe mortalities. RTGE has been reported to Cefas on a few farms in England and Scotland since 2003. This disease was investigated further under Objectives 2 and 3.

SID 5 (2/05) of 27

01.6 Shewanella putrefasciens (also described as Achromobacter putrefasciens, Pseudomonas putrefasciens and Alteromonas putrefasciens). This was isolated on a number of occasions by CEFAS microbiology diagnostic function staff from moribund carp submitted for bacteriology investigation by the Fish Health Inspectorate, in conjunction with the EA. There is increasing evidence of its pathogenicity, with incidences of disease in carp and rainbow trout attributed to S. putrefaciens reported from Poland (Kozinska and Poekala, 2004)

01.6 Fungal infections in salmonids (including brown trout) and recently vaccinated salmon.

01.7 A ‘saddleback disease’ in salmon parrA Chryseobacterium sp. has been implicated in this condition. This was investigated further

under FC1151 (Objective 3.).

01.8 Red Mark SyndromeA skin condition of rainbow trout, Red Mark Syndrome, emerged in 2005 in England and Wales. This was investigated further under FC1151 (Objective 3.4).Objective 01 General conclusions Heavy losses due to many of these conditions were repeatedly reported by CEFAS FHI, fish vets and farmers over the period of the project. An apparent, perceived, lack of efficacy of Pyceze, the new alternative to Malachite green was also reported. This finding has been reported to the product manufacturer. Cefas FC1151 project staff have also been involved in a CARD funded project (PN 0117) to help develop alternative Saprolegniasis treatments, demonstrating how FC1151 has partly enabled other important research to proceed.Of relevance to the UK, a new pathogen was described in Norway in salmon. The disease was responsible for losses in freshwater fish and was caused by Rhodococcus erythropolis, a Gram positive bacilli. Contacts were established with the group that described the pathogen and the disease and an isolate obtained.L. garvieae and other Gram positive cocci in farmed fish were received from European, researchers as a result of contacts made by Cefas scientists at international conferences. A good level of communication was maintained with all relevant groups within Cefas (i.e. FHI and epidemiology) to ensure a rapid dissemination of information. During the lifetime of the project, the speed with which Cefas has been able to respond to emerging diseases in general, as well as those caused by bacteria and fungi, has been further improved by the creation of project FC1166; ‘Characterisation and Pathogenesis of fish and shellfish emerging diseases’. Under FC1166, there are regular meetings between the multidisciplinary project staff and Defra project manager to review all emerging diseases, including those caused by bacteria and fungi. It is not always obvious what is causing a particular emerging disease (e.g. Red Mark Syndrome in rainbow trout that is described below under Objective 3.4), thus it is important that specialists from a range of different disciplines assess any new threat.

Objective 02. Objective as set out in the contract: To develop diagnostic capabilities for emerging bacterial and fungal pathogens of fish and shellfish

Extent to which objective 2 has been met. This objective as a whole was met over the course of the project, with greater progress made in some areas then in others.

SID 5 (2/05) of 27

Objective 02: Details of the methods used and the results obtained, including statistical analysis (where appropriate) and a discussion of the results and their reliability.Work completed under FC1151, in conjunction with FA001, focused on a range of diseases.

02.1 Identification of Gram positive cocci (GPC) pathogenic for fish

02.1.1 INTRODUCTIONAvailable methods generally depend upon the characterisation of the phenotypic and biochemical patterns of each isolate followed by a comparison of these profiles with published references. Biochemical variations amongst strains isolated from different fish species or geographical areas, methodological variations between laboratories and, to some extent, a lack of accurate data have made the use of published profiles and miniaturised biochemical tests such as the Rapid ID 32 Strep system (Biomérieux, France) unreliable for accurate identification of fish pathogenic GPC. Because of the limitations of classic bacteriology identification kits, many workers have turned to the development and validation of molecular biological tools such as 16S rRNA sequencing, or the polymerase chain reaction (PCR), for the identification of such pathogens. Many of these tools have now been developed and are being used to identify these organisms accurately. These techniques are however expensive and time consuming and, although powerful, are not available in all fish diagnostic laboratories. As part of FC1151, we investigated the use of Biolog’s Microlog System (Biolog, USA) to identify and characterise some of the Gram-positive cocci listed above together with other GPC that may be markers of environmental contamination. Unlike other secondary tests, the Microlog System can recognise over 4x1028 possible metabolic patterns by testing the ability of test bacteria to use as many as 95 different carbon sources. The test results in a pattern or fingerprint, which is claimed to be unique to the test bacterium and readable visually. The fingerprint data having been fed into Microlog software, the manufacturer claims that a bacterial identification may be possible within 4 to 6 hours of inoculation of the Biolog plate. The results obtained for the identification of a number of pathogenic and non-pathogenic GPC using the Microlog system were compared to that obtained using Rapid ID 32 Strep galleries (Biomérieux, France).

02.1.2 MATERIALS AND METHODSBacterial strains and culture conditions: Twenty one field isolates and two reference isolates of L. garvieae were tested in this study. A number of reference isolates of other Gram positive cocci either pathogenic or non-pathogenic to fish were also included in the study for comparative purposes. The identity of all isolates was confirmed prior to the start of the study by 16S rRNA gene sequencing (data not shown) and comparison of the obtained sequences to the bacterial 16S RRNA gene sequences submitted to GenBank and EMBL databases using the BLAST search program available at the UK HGMP Resource Centre, Hinxton.

RAPID ID 32 STREP test: The Rapid ID 32 Strep is a standardised system for the identification of streptococci and related organisms which uses 32 miniaturised enzymatic tests each containing a dehydrated test substrate. When inoculated with a bacterial suspension the enzymatic substrates are rehydrated and the metabolic end products produced during incubation detected. In the conventional method, all bacterial isolates were grown on Columbia sheep blood agar (Sigma, USA) supplemented with sheep blood (Oxoid Ltd, UK) for 18-24 hours at 37°C. A suspension of the bacterium was then made from well isolated colonies in sterile distilled water to a McFarland standard of 4. Each test well was then inoculated with 55µl of the suspension. A small amount was also added to a Tryptone soya agar (TSA, Oxoid) plate to act as a purity

SID 5 (2/05) of 27

check. Both were then incubated at 37°C, and the results recorded after 4 to 4.5 hours incubation. For the purpose of the modified method, all bacterial isolates were grown on Columbia sheep blood agar, but the agar plates were incubated at 25°C for 24-48h. The optical density of the bacterial suspension was spectrophotometrically adjusted to an optical density (OD) of 0.8 at 580 nm and the incubation temperature for the test strip was lower at 25°C. Results were recorded after 4 to 4.5 hours incubation. Bacterial identification was achieved using the Biomérieux identification software (API database V 2.0). The bacterial isolates were considered to be correctly identified when the API software gave an “Excellent” or “Very Good” identification result. Any other result (“Doubtful” Lactococcus garvieae profile or complete misidentification) was considered as a failed identification.The profiles of all isolates tested using the modified procedure were also compared to the standard Lactococcus garvieae profile determined by Ravelo et al. (2001).

Biolog System: The tests were performed using the BIOLOG GP2 Microplate system, which is described as a standardised system for the identification and characterisation of a broad range of Gram positive bacteria by their ability to utilise or oxidise compounds from a pre-selected panel of carbon sources. The system uses 95 miniaturised biochemical tests each containing a different dehydrated carbon source and a redox dye. When inoculated with a bacterial suspension (in a gelling' inoculating fluid) the tests are rehydrated and during incubation the bacterial respiratory burst oxidises the dye, which then becomes purple. The ability of the bacteria to utilise the various carbon sources yields a characteristic pattern of purple wells. To characterise the test isolates using the Biolog system, the bacteria were subcultured twice onto BUG agar with 5% sheep blood and incubated under optimum growing conditions (previous work indicated a temperature of 30°C for 20±2h). After the second incubation period, three drops of the anti-capsular agent sodium thioglycolate were added to a tube of sterile "gelling" Gram positive inoculating fluid and a suspension of the bacterium prepared to a 20±2% transmittance level. Each test well of the GP2 Microplate was then inoculated with 150µl of the bacterial suspension, a small amount also added to a TSA plate to act as a purity check. Both were then incubated at 30°C. The colour pattern of each plate was read by the naked eye against the colour of the control well after 4 to 6 and 16 to 24 hours incubation.. Identification was achieved using Biolog's MicroLog 1 software.

02.1.3 RESULTSTable 1 presents the results of our API RAPID ID 32 STREP tests using both the conventional and the modified methodologies. As shown, none of the test methods performed on our 23 Lactococcus garvieae isolates gave a reliable identification. Only one of the reference isolates was correctly identified using the conventional or modified test procedure and the API software. Less than 60% of the field isolates were correctly identified using the same methods. Comparison of the test isolate profiles to Ravelo et al. standard Lactococcus garvieae profile failed to identify any of the field or reference isolates. These methods also failed to correctly and reliably identify other GPC, which may be potential fish pathogens or environmental contaminants. For Biolog, pate reading after only 4-6h incubation proved difficult. After such a short incubation period, 79% of the Lactococcus garvieae isolates and 30% of the other GPC were however correctly identified. The rest of the isolates were either not identified or misidentified. After 16-24h incubation (Tables 2-3), all tested Lactococcus garvieae isolates were correctly identified. The test also allowed us to correctly identify most of the other GPC. Lactococcus piscium was unable to grow on BUG agar making its diagnosis and identification impossible with this technique. Finally the test also wrongly identified Streptococcus iniae as Lactococcus garvieae in one instance only.

SID 5 (2/05) of 27

Table 1 Identification of Lactococcus garvieae and other fish pathogenic and non-pathogenic Gram positive bacteria using API RAPID ID 32 STREP. Number of correctly identified isolates using either the API database or Lactococcus garvieae profiles given by Ravelo et al. (2001).

consideration.

02.2. Culture and Diagnostic of Piscirickettsia salmonis and Rickettsia-like organisms :

Work was done to ensure that CefasWeymouth has the capability to diagnose these emerging pathogens. The type strain LF-89 was purchased from ATCC and a Scottish wild isolate was received from Dr. H. Birkbeck at Glasgow University. P. salmonis is notoriously difficult to culture, passage and store. It must be grown in cell culture without the addition of antibiotics and is reputed to lose 99% of its viability on each freeze thaw cycle. Contamination problems within the cell lines initially slowed down progress with this objective. Purchase of new cell lines and strict adherence to aseptic technique overcame initial problems. Two different cell lines were compared for their efficiency at growing P. salmonis - the normally used Coho Salmon Embryo-214 (CHSE) and an insect cell line, SF-9, which has been reported by colleagues at Glasgow University to yield higher titres of P. salmonis and be more sensitive to Rickettsia infection. We did indeed find that P. salmonis grows more rapidly and to a higher titre in SF-9 - we achieved a titre of 108 TCID50/ml which was 1 log greater than in CHSE-214 cells. However we found it much more difficult to track the progress of the infection in SF-9 as there was not the distinctive cytopathic effect (CPE) progression as displayed in CHSE-214.

The storage of viable P. salmonis at various temperatures was investigated. Storage was attempted at 4°C, –80°C and liquid nitrogen. Survival at 4°C gave us better results than expected and reported in the literature (no drop in titre in the first week but a significant drop thereafter and below detection limit after 3 weeks). Storage in liquid nitrogen and at –80°C gave us excellent long-term survival results with titres of 104 TCID50/ml regularly achieved after 18 months. In terms of diagnostic methods: Atlantic salmon were successfully challenged with P. salmonis. Both external and internal clinical signs were observed and photographs taken. Samples were taken for histology and added to the Registry of Aquatic Pathology. An Immuno Fluorescence Antibody Technique (IFAT) has been developed for identification of the pathogen from tissue cultures. It was applied to tissue imprints from infected fish and allowed clear visualisation of the pathogen in infected fish with no fluorescence observed in control fish. Re-isolation of P. salmonis was attempted from infected tissues by culture in CHSE cells. However all attempts resulted in bacterial contamination. Cefas is now able to recognise clinical signs of Piscirickettsiosis and identify P. salmonis from both Gram and IFAT stained sections and smears. Infected tissue has been frozen and stored for the possible development of molecular methods in the future.

02.3. Identification of Candidatus arthromitus .

C. arthromitus associated with RTGE was identified as a potential emerging pathogen in England in Summer 2003. The aquaculture industry was keen that tools be developed to try and identify the early presence of this unculturable bacterium in the fish gut, so that effective control methods could be put in place to try and prevent the development of the enteric disease. The bacterium being extremely fragile and denaturing very rapidly in gut samples, it was proposed that a method to identify the presence of the bacterial endospores as opposed to the presence of the bacterium itself should be developed. In the absence of Candidatus material, a number of spore staining protocols were tested using Bacillus as a model organism. The best results were obtained using Ziehl Neelsen and modified Ziehl Neelsen staining procedures. These were applied to a few samples but the absence of C. arthromitus material prevented any extensive testing and use of the test protocol in the field. Attempts to identify the causative agent using culture-independent molecular methods did not prove successful. (DNA of bacterial origin was PCR amplified from infected fish gut samples using eubacterial primers and cloned into E. coli). The cloned inserts were sequenced and compared with GenBank deposited 16S rRNA gene sequences, with none of the cloned sequences showing homology to Segmented Filamentous Bacteria derived sequences.

02.4. Identification of emerging strains of Yersinia. ruckeri :

A number of Y. ruckeri field isolates, supposedly of serovar 02, and stored in Cefas bacterial collection were tested using API 20E and serological tools. All but one of these serovar 2

SID 5 (2/05) of 27

isolates were confirmed as serovar 2 (Microtek rapid agglutination test) and shared very similar API20E profiles (1305500 or 1307500). At the same time, field Y. ruckeri isolates isolated between 2002 and 2005 and stored in Cefas bacterial collection and at FRS Aberdeen were also tested. All but one of them were confirmed as belonging to the Hagerman group (serotype 1) using both Microtek kit and anti-sera raised in house by agglutination testing. They also shared similar API20E profile (1/5 1/3 7100), this profile being distinct from that of serovar 2 strains. The field isolate (03031 – PM 10238) originated from a trout farm with numerous vaccination problems.

A range of Y. ruckeri isolates recovered from both salmon and rainbow trout were firstly compared using Biolog GN. This technique was not found to be suitable for discriminating these organisms, possibly reflecting the biochemical uniformity previously noted for Yersinia ruckeri (Austin and Austin, 1999).

An initial attempt to examine a range of strains using Pulsed Field Gel Electrophoresis (PFGE) was also carried out. PFGE, is recognised as the ‘gold standard’ for typing of bacteria (Maslow & Mulligan 1996, Streulens 1998) and is the current method of choice for typing nosocomial and community acquired human pathogens (Tenover et al 1997). Despite only detecting differences in <0.01% of the chromosome, important genomic reorganisation caused by duplication, mutation, deletion or insertion are apparent as a change in fragment size or number. It also demonstrates excellent reproducibility and resolution. Thus in comparison with other methods it has a high level of discriminatory ability and most systematic or partially systematic studies of sub typing performance include comparisons with PFGE.

We compared seven Y. ruckeri isolates, including strains recovered from Atlantic salmon, the Hagerman 01 type strain and an example of an emergent Y. ruckeri strain affecting rainbow trout in England and Wales. Interestingly the pulsotype for the emergent rainbow trout strain was more similar to the Hageman 01 strains then to the other strains tested against, even though, as discussed above it is serologically and biochemically distinct (Figure 1)

Figure 1. PFGE profiles of Y. ruckeri. Lane 1, Hagerman O1 ATCC 2194, Lane 2, emergent Y. ruckeri strain isolated from rainbow trout. Lane 3, Y. ruckeri 263, O2 serotype isolated from Atlantic salmon. Lane 4 Y. ruckeri 176, O2 serotype isolated from Atlantic salmon. Lane 5, Y. ruckeri 99178, 02 serotype. Lane 6, Y.ruckeri

SID 5 (2/05) of 27

MM 1 32 M4 5 76

03051, 01 serotype isolated from rainbow trout. Lane 7, Y. ruckeri 78029, 02 serotype.

M = molecular size standard.

Future Work

Further work is planned to investigate how Y. ruckeri has evolved to circumvent currently available control measures. Defra has agreed to the funding of a sandwich studentship for the period 2006-2007. The work will be done in collaboration with Dr Robert Davies, from Glasgow University who are planning to supply the student. Dr Davies, an expert on Y. ruckeri identification and pathogenesis, has agreed to supervise the student involved and also potentially allow the student to do some preliminary work in his laboratory before the project starts.

02.5. Identification of common fish pathogenic Vibrio species. Work was undertaken in the project to better equip Cefas Weymouth with Vibrio identification tools. The correct identification of Vibrio species pathogenic to fish is reputedly problematic but work has been done under this heading to investigate the merits of combined Biolog GN and 16S rRNA sequencing identification tools for the more common Vibrio species. Preliminary studies were undertaken by a French veterinary student during a work placement at Cefas Weymouth and supervised under FC1151 (Gaultier, 2006). A MRes student from the University of Plymouth continued the work, this time looking at Vibrio spp. affecting seahorses cultured in UK zoos and aquaria (Amey, 2005).As an outcome of these studies, Cefas is now in a position to reliably identify Vibrio spp. Using a range of techniques.

02.5.1 BACTERIAL INFLUENCES ON SEAHORSE (HIPPOCAMPUS SPP.) SURVIVALWild seahorses are currently collected in large numbers for use in traditional medicine, for ornamental display and as a curio. To reduce pressure on wild stocks, the Convention on International Trade in Endangered Species (CITES) of Wild Fauna and Flora (CITES), prohibited the capture or movement of seahorses by adding them to Appendix II in 2002. However, demand for seahorses remains high, which has led to numerous captive breeding initiatives by public aquaria and the recent development of seahorse farms.The UK has been heavily involved in these initiatives with at least five zoos and aquaria operations with active seahorse captive breeding initiatives. Anecdotal reports suggested that high mortalities during the early life stages are commonly reported, as for farming of other marine finfish species. It is possible that these high and variable mortalities could have been caused by bacteria, particularly as farmers and aquarists often have to use antibiotics to obtain good survivals. Very little is known about the bacterial flora of seahorses in either the wild or captivity though.With the cooperation of three organisations, The National Marine Aquarium in Plymouth, a farmer in Wales, and Chester Zoo, a preliminary histopathological and bacteriological survey of healthy and moribund captive seahorse juveniles and young adults (mainly Hippocampus kuda sp.) was undertaken by a self-funded MRes student from the University of Plymouth, with limited support from F1151 (supply of bacteriological media and other consumables). 178 bacterial isolates recovered from seahorses from the three seahorse sites were grouped based on nine phenotypic characters (colony morphology, pigment production, Gram, catalse activity, cytochrome oxidase activity, growth on Thiosulphate citrate sucrose bile salt agar (TCBS) and growth at 4 °, 22 and 37 °C. From this 76 typical isolates were biochemically characterised using BIOLOGGN. (BIOLOGGN is similar to the BIOOGGP system described in more detail under Objective 2.01 but designed for the identification of Gram negative organisms). Organisms were then clustered on the basis of their BIOLOGGN profiles and placed into eight distinct phenons. Initial indications are that the large majority of the bacteria that could be recovered from seahorses on conventional media belong to the Genus Vibrio (70/76 characterised isolates were

SID 5 (2/05) of 27

Vibrio sp.). Likely species recovered included Vibrio splendidus and Vibrio alginolyticus, species commonly recovered from UK marine finfish hatcheries (Verner – Jeffreys, 2003). Phenotypic analysis indicated that many of the organisms isolated from different rearing centres cited at different geographic centres were highly similar. 11/76 haemolytic isolates recovered from moribund farmed seahorse juveniles formed a distinct cluster when characterised using BIOLOG GN, suggesting they were highly similar. It is possible these organisms are examples of a Vibrio species or biotype that is pathogenic towards seahorses, however further work is required to confirm these initial findings.Histological techniques for preserving and staining tissue sections from seahorses were successfully optimised. Pathologies were not generally noted in processed samples, although focal inflammatory lesions containing accumulations of Gram positive bacteria were identified in musculature, liver and kidney of a moribund H. abdomanalis specimen from the Plymouth Marine Aquarium. It is possible that observed mortalities in farmed seahorses could have been caused by these bacteria.Further work is required to, firstly, confirm the identities of the partially-characterised isolates using molecular tools (e.g.16S rRNA gene sequencing of phenon representatives) and, secondly, to further characterise the organisms recovered from the apparently diseased seahorses. The project as a whole identified that, if efforts are to be made to farm these particular species, further work will likely be necessary to both identify the diseases the farmed species are likely to be affected by, as well as develop appropriate control strategies.

02.5 Development of 16S rRNA gene based identification techniques.Identification of bacteria based on 16S rRNA gene sequence analysis is a well-established and reliable method. FC1151 was used to establish and validate a 16S rRNA gene based identification protocol in the Weymouth Laboratory for both Gram negative and Gram positive organisms. An example of the successful application of the developed protocol was the identification of Shewanella putrefaciens, isolated from moribund carp, confirming the species identity of the Gram positive cocci described earlier (Objective 2.1), identifying the Chryseobacterium isolate recovered from moribund Atlantic salmon described under Objective 3.2.. as well as identifying a range of motile and non motile Aeromanas spp.

Objective 03. To investigate and assess the impact of new or emerging bacterial and fungal pathogens of fish and shellfishA range of diseases were investigated over the course of the project.

03.01.Susceptibility of selected freshwater course and salmonid fish species to Lactococcus garvieae03.1.1 INTRODUCTIONLactococcosis, caused by the bacterium Lactococcus garvieae, has been reported in farmed Rainbow trout in many European countries including Italy, Spain, France, the United Kingdom and Portugal. This disease poses a significant threat to trout farming with mortality levels reaching 90% observed at water temperatures above 16-18ºC (Pereira et al., 2004). Despite its severity, there is little information on L. garvieae virulence in other freshwater fish (salmonids and non-salmonids) which may be farmed alongside rainbow trout or reside in the vicinity of trout farms affected by lactococcosis outbreaks. These species could potentially be exposed to the pathogen or to infected or carrier fish in natural circumstances. Such a case occurred in 2000 when the first UK outbreak affected a large trout farm operating on a freshwater reservoir and nature reserve with natural populations of coarse fish. In a series of experiments we investigated the susceptibility of three salmonid species (Atlantic salmon, brown trout and grayling) and seven cyprinid fish species (carp, tench, rudd, barbel, chub, dace and roach) to L. garvieae. We first used intraperitoneal (i.p) injection of a fairly high concentration of the bacterium as a challenge method to assess the virulence of the organism. Secondly, we used a cohabitation challenge to better mimic a natural infection route.

SID 5 (2/05) of 27

03.1.2 MATERIALS AND METHODS Bacterial isolateA L. garvieae isolate from the 2000 UK outbreak was used in all trials. Its identity was confirmed using standard biochemical and molecular techniques and has since been stored at –80ºC. When required, the bacterium was cultured in TSB and re-suspended in PBSa to the desired concentration.

ChallengeInjected fish were anaesthetised using MS222 and ip injected with 0.1 ml of L. garvieae suspension. Carp, tench, rudd, barbel, chub, dace, roach and rainbow trout (RT) weighing between 11 and 35g were each injected with 1.09x106 colony forming units (cfu) /fish. Atlantic salmon, grayling, brown trout and RT weighing between 4 and 22g were each injected with aproximately102, 104 or 107 cfu/fish.Cohabitant fish were kept in the same tanks as rainbow trout ip injected with L. garvieae (direct cohabitation) or received outlet water from tanks containing L. garvieae injected fish (indirect cohabitation).Rudd, chub, roach and RT weighing between 7 and 22g were directly cohabited with RT injected with 8.5x103 cfu/fish. Atlantic salmon, grayling, RT and brown trout between 4 and 22g were directly cohabited with RT ip injected with approximately 104 cfu/fish. Brown trout, grayling and RT between 4 and 22g were indirectly cohabited with RT injected with 1x104

cfu/fish.

Post challengeAll challenged fish were sampled (by inoculation of kidney material onto TSA) to check for presence of L. garvieae. Specific mortality and carrier status for each species was calculated. Any mortalities from which L. garvieae was not recovered were discounted from calculations.

03.1.3 RESULTSCyprinid fishThe susceptibility of cyprinid fish to L. garvieae, and carrier status amongst surviving fish are summarised in Table 4. ‘Carrier status’ was determined on the basis of reisolation of viable L. garvieae from the haed kidney on TSA. Moribund and dead fish showed only a few signs of infection, including changes in swimming behaviour (lethargy, loss of orientation and surface swimming) and at times tail and fin erosion. Slight exophthalmia was observed in tench, roach and dace and internal haemorrhages observed in tench. None of the fish exhibited gross signs of lactococcosis at the end of the trial.

SalmonidsThe results of exposure of salmonid fish to L. garvieae by ip injection are presented in Table 5. Comparative susceptibility of these fish to different doses of bacteria is shown in Figure 2. The results of exposure of salmonid fish to L. garvieae by cohabitation are shown in Table 6.All dead and moribund salmonids showed classic lactococcosis signs, including exophthalmia, melanosis, internal haemorrhages, ascitis and enlarged spleen. All surviving fish, however, looked healthy.

03.1.4 CONCLUSIONSWith a LD50 at D20 below 2x102 cfu/fish, the first UK L. garvieae isolate is at least as virulent for rainbow trout as its Portuguese and Italian counterparts for which LD50 of 101-102 CFU per fish were reported (Ghitthino et al., 1998; Pereira et al., 2004). The L. garvieae isolate is highly virulent for grayling. Other salmonids such as Atlantic salmon and brown trout are susceptible to the pathogen and can develop the diseaseCourse fish such as carp, tench, barbel, chub, dace and roach can all succumb to L. garvieae infections. Rudd populations appeared relatively resistant with only 1 fish dying of

SID 5 (2/05) of 27

lactococcosis in the whole of our trials.Covert L. garvieae infections can occur in fish that have survived a lactococcosis outbreak. Similar findings had been observed by Muzquiz et al. (1999). Our direct and indirect cohabitation challenges demonstrated that healthy carrier fish could be detected in all salmonid and cyprinid species tested apart from rudd.The proportion of carrier fish within a population previously exposed to L. garvieae was significant, particularly amongst salmonid fish (>18%). Carrier fish can act as a reservoir of the bacterium which may be shed back into the environment when conditions are favourable and transmit the infection to other fish (Austin and Austin, 1999). Carrier fish are known to play a major role in the epizootiology of other bacterial diseases such as furunculosis (Hiney et al., 1997). Our findings, along with data on the relative susceptibility of various fish species, are therefore important for the management of surviving stocks and predicting the impact of lactococcosis on wild and farmed fish species.

Table 4: Mortality and carrier states in selected cyprinid fish exposed to Lactococcus garvieae by intra-peritoneal injection or cohabitation with infected fish ((carrier state determined as % surviving fish L. garviae reisolated from)

Challenge method

Fish species Number of fish at D01

Specific mortality

(%)

Number of survivors at

DF2

Carrier rate (% of surviving

population)Intra-peritoneal injection (1.09x106 CFU/fish)

Tench 29 6.9 27 22.2Carp 30 3.3 29 31.0Rudd 30 0 30 3.3Bream 31 3.2 30 40.0Chub 30 13.3 26 11.5Dace 31 19.4 25 20.0Roach 29 10.3 26 19.2Rainbow trout 10 100 0 -

Cohabitation with infected rainbow trout (8.5x103 CFU/fish)

Chub 33 3.0 31 3.2Rudd 30 3.3 28 0Roach 22 4.5 20 5.0Rainbow trout 30 76.7 7 57.1

Note 1: D0 = day of the intra-peritoneal injection challenge

Note 2: DF = day of the termination of the trial (respectively D20 for ip injection and D27 for cohabitation trials)Table 5: Mortality and carrier states in salmonid fish exposed to L. garvieae by intra-peritoneal injection.

Challenge method Fish species Number of fish at D01

Specific mortality

(%)

Number of survivors

at DF2


population)Ip injection

2.0 102 CFU/fish Atlantic salmon 15 13.3 13 84.6Grayling 20 65 7 85.7Rainbow trout 20 100 - -

2.0 104 CFU/fish Atlantic salmon 15 6.7 14 85.7Grayling 20 90 2 100Rainbow trout 20 100 - -

2.0 107 CFU/fish Atlantic salmon 15 40 9 100Grayling 20 100 - -Rainbow trout 20 100 - -

Ip injection6.1 101 CFU/fish Brown trout 5 20 1 1006.1 103 CFU/fish Brown trout 6 0 6 16.6

Rainbow trout 5 80 1 06.1 106 CFU/fish Brown trout 5 20 4 100

SID 5 (2/05) of 27

Table 6: Mortality and carrier states in salmonid fish exposed to Lactococcus garvieae using a direct or indirect cohabitation challengeChallenge method Fish species Number of

fish at D01Specific mortality

(%)

Number of survivors

at DF2


population)Direct cohabitation with Rainbow trout infected by ip injection (1.6 104 CFU/fish)

Atlantic salmon 57 0 54 18.5Grayling 60 40 22 54.5Rainbow trout 30 93.3 1 100

Direct cohabitation with Rainbow trout infected by ip injection (1.0 104 CFU/fish)

Brown trout 60 0 60 76.6Grayling 57 40.3 34 35.3

Indirect cohabitation with Rainbow trout infected by ip injection (1.0 104 CFU/fish)

Brown trout 60 1.7 57 31.6GraylingRainbow trout

5930

37.386.7

334

18.225

Note 1: D0 = day of the intra-peritoneal injection challengeNote 2: DF = day of the termination of the trial (D24)

03.2. Investigations into the causes of a new severe chronic skin condition affecting Atlantic salmon ( Salmo salar l.) Pre-smolts

03.2.1 INTRODUCTIONIn late summer 2002 Atlantic salmon pre-smolts (av. weight 39g) recently transferred to the Cefas Weymouth experimental facilities, where they were held in freshwater, began to show signs of an unusual disease. The fish, obtained from a major smolt supplier had been checked on their arrival at the laboratory and confirmed to be free of common fish pathogens. Part of the stock was kept as naïve fish while others were vaccinated or mock-vaccinated (i.p.) using PBSa) or commercial test vaccines. All three groups were subsequently held separately in freshwater at around 12°C in 300l tanks, at a stocking density not exceeding 26 kg/m3. The fish were fed a standard commercial salmon feed at the maintenance rate of 1% BW/d. From 3 to 5 days following i.p. injection, mortality started in all injected groups. Dead fish showed haemorrhaging between the pectoral fins and the gills, a “saddleback” type lesion around the eroded dorsal fin, severe erosion of pectoral fins, gill necrosis and on most moribund

SID 5 (2/05) of 27

Figure 2: Comparative susceptibility of salmonid fish exposed to various doses of Lactococcus garvieae by intra-peritoneal injection

0

10

20

30

40

50

60

70

80

90

100

110

0 4 8 12 16 20 24

Days post- challenge

Cum

ulat

ive

mor

talit

y (%

)

Brown trout - 6.1 10^6 CFU/fish Brown trout - 6.1 10^3 CFU/fish Brown trout - 6.1 10^1 CFU/fishRainbow trout - 2.0 10^7 CFU/fish Rainbow trout - 2.0 10^4 CFU/fish Rainbow trout - 2.0 10^2 CFU/fishAtlantic salmon - 2.0 10^7 CFU/fish Atlantic salmon - 2.0 10^4 CFU/fish Atlantic salmon - 2.0 10^2 CFU/fishGrayling - 2.0 10^7 CFU/fish Grayling - 2.0 10^4 CFU/fish Grayling - 2.0 10^2 CFU/fish

and dead fish fungal growth on the dorsal and pectoral fins and around the gills. A number of treatments (inc. salt, hyamine, chloramine T) were administered. Although an immediate slight improvement was noticed with significantly reduced mortality being recorded in the days that followed hyamine and chloramine T treatments, mortality soon resumed and overall about 80% of the original stock succumbed over a 6-month period. Increasing the temperature to above 15°C appeared to have no effect.Moribund fish were sampled in an effort to identify the origin of the problem. Transmission trials were also instigated to establish whether the condition was infectious. The severity of the condition differed between the different groups in the order vaccinated fish > mock vaccinated > naïve fish. Through industry contacts we became aware that a similar problem was being encountered in the field in pre-smolt salmon following i.p. vaccination with multivalent oil-adjuvanted vaccines.

03.2.2 RESULTS OF DISEASE INVESTIGATIONSThe effects of several vaccines were explored and it became clear that whilst vaccination would result in a quicker and more virulent expression of the condition, the effect was to disclose a pre-existing condition. Moribund fish showed signs of a ‘saddleback’ lesion surrounding an eroded dorsal fin. The skin appeared spongy with an increased quantity of mucus, raised or missing scales and petechial haemorrhages. Presence of external fungal hyphae was observed on most fish at death. Haemorrhaging was present on the skin, particularly between the pectoral fins. Extensive areas of necrosis were observed on the gills and the spleen was often enlarged. A range of possible aetiologies were investigated. No viruses were detected. The Saprolegnia spp. isolated from the gross lesions were primarily S. diclina rather than S. parasitica. A range of bacteria were isolated from fish at different stages of the condition. These included Lactococcus sp. and Flavobacterium sp., which were present in moribund animals.

SID 5 (2/05) of 27

Figure 3 and Figure 4 Atlantic salmon affected by ‘saddleback’ syndrome. Note extensive erosion of skin and associated fungal growth on lesions and pectoral fins .

F. psychrophilum was ruled out early in the investigation. A Chryseobacterium sp. was, however, isolated consistently from skin and viscera of affected animals. The organism was identified by a combination of molecular and biochemical methods (16S rRNA gene sequencing, API 20E and the results of primary tests).

Figure 5. Internal clinical signs of an Atlantic salmon affected by the condition. Note petechial haemorrhaging over ventral surface and internal organs.

03.2.3 VIRULENCE TRIALSFish from the infected stock showing typical external clinical signs were cohabited with 20 naive fish from a separate healthy stock of salmon. Ten of these fish were ip vaccinated with a multivalent oil-adjuvanted vaccine. The fish were kept in freshwater at a temperature around 12-13°C for up to 3 months during

which morbidity and mortality were recorded and investigated. Mortalities were first recorded in the infected stock (20% - D9), followed by the vaccinated fish (20% - D19) and the naive fish (10% - D36). All dead fish showed the clinical signs described above and overall, almost all of the naive, vaccinated and infected stock showed some clinical signs (from slight to severe) during the course of the investigation. This indicates that transmission was successful, that the condition is of infectious origin and that the causative agent is waterborne.

03.2.4 CONCLUSIONSThe disease was demonstrated to be infectious. No parasite or virus was detected but a number of bacterial and fungal isolates were recovered from sacrificed moribund fish. Aeromonas sobria, A. hydrophila, Acinetobacter sp. and P. fluorescens have been reported to cause disease in wild or farmed fish (Ahne et al., 1982 ; Toranzo et al., 1989 ; Dierckens et al., 1998 ; Roald & Hastein, 1980) however the conditions they are responsible for are very different from the one reported here. Saprolegnia sp was found on the skin and gills of salmon showing severe clinical signs. The fungus may well play an important part in the latest stage of the disease but we do not believe that it is responsible for its onset as the condition was only associated with particular stocks of fish.. The exact cause of the disease remains uncertain although it is believed that it may have been caused, at least in part, by one of the many “Flavobacterium” type bacteria (here identified as Flavobacterium sp. or Chryseobacterium sp.) that we repeatedly recovered from the skin and internal organs of infected fish. An F. psychrophilum or F. columnare aetiology has been ruled out. However, attempts to confirm the virulence of isolated Chryseobacterium were unsuccessful. This does not however prove the organism was not responsible; it is very possible that the isolates tested had lost virulence in culture. This is a common problem with studies involving F. psychrophilum and related organisms.The condition appeared to be more severe in vaccinated fish but our investigations to date have failed to provide sufficient information to fully support this. A condition similar to the one described here is known to be present only on a small number of farms in the UK. The disease is however difficult to diagnose and with moribund fish developing a secondary fungal infection, it is possible that many of the recent fungal problems observed in trout and salmon farming in the UK (in particular the so called “post-vaccination” syndrome) were indeed secondary to the disease described here.

03.3 Virulence of Candidatus arthromitus in rainbow trout

SID 5 (2/05) of 27

Figures 3-4 Atlantic salmon affected by ‘saddleback’ syndrome. Note extensive erosion of skin and associated fungal growth on lesions and pectoral fins.

03.3.1 WORK COMPLETEDIn vivo trials were planned to try and investigate the transmission of this unculturable bacterium. An amendment to our Home Office license was obtained to carry out such work. The trials, relying on the availability of diseased fish from farms affected by the disease, did not take place until September 2005 due to a lack of suitable fish material. A study was then run. One trial involved feeding gut material from allegedly RTGE-affected fish to naïve fish. The other was a conventional direct cohabitation trial. The disease was not successfully transmitted, the most probable reason being that the disease had recessed between it being reported to Cefas (early August) and Cefas study staff gaining access to the farm to obtain ‘infected’ fish for transport back to Cefas Weymouth Laboratory to run transmission experiments. High mortalities were observed in the test fish groups but these were attributable to infection by Aeromonas salmonicida subsp. salmonicida, which was re-isolated from mortalities (along with clinical signs, including liquefaction of the internal organs). It was possible that this was facilitated by the high water temperatures used for the study (19 °C), as A. salmonicida subsp. salmonicida is not generally highly virulent towards rainbow trout, except at such high temperatures.

03.3.2 PLANNED FUTURE WORKAlthough FC1151 has terminated, work into emerging diseases continues at Cefas under different projects (e.g. FC1166). It is proposed to complete the characterisation of samples already collected from RTGE-affected farms by phylogenetic comparison of SFB-associated DNA in those samples. Recognising the importance of this disease, the British Trout Association and CARD have recently funded a PhD studentship through the University of Stirling to investigate RTGE and develop methods for its control. It is envisaged that the work undertaken will be complimentary to this other funded work and any information Cefas generates will be shared with University of Stirling researchers so we can build up a better general picture of the causes, epidemiology and associated risks of this disease in the UK as a whole. Informal discussions have already been held between researchers from both Cefas and Stirling to ensure this process is transparent and efficient. Among the important factors that have not so far been addressed are the potential risks of transmission of this disease to wild salmonid and other fish species.

03.4 Studies on Red Mark Syndrome

Red Mark Syndrome (RMS) is a transmissible disease of rainbow trout, characterised by the appearance of multiple ulcerated skin swellings, of varying intensity, on the flanks of affected

SID 5 (2/05) of 27

fish. It shares some similarities with Strawberry Disease, not least that the infectious agent responsible has yet to be definitively identified. However, there are enough differences from Strawberry Disease, in terms of both its epidemiology and pathological effects, to regard RMS as a separate condition. The condition causes losses to farmers in that affected fish are down graded at harvest. There are also some reports of increased losses of RMS-affected stock during grading and other stressful procedures.

03.4.1 HISTORY OF THE DISEASEThe condition was first noted in Scotland in 2004. In early 2005, the condition was diagnosed for the first time in fish farmed in England. Farmers in both Scotland and England report that the disease is prevalent at low temperatures (less than12 °C) and early signs of the condition can include severe scale loss prior to the emergence of the characteristic external lesions.Recently there have been reports of an RMS-like condition in Wales, with evidence of spread from an infected farm to at least two other farms.

03.4.2 DISEASE INVESTIGATIONSInvestigations of the two English farms suffering from RMS were instigated under FC1151. Epidemiological analysis indicated that the condition on both farms originated in stocks of fish sourced from the same Scottish hatchery. This hatchery had, itself, suffered RMS outbreaks in the past. There were also indications that the condition had spread from the imported, infected, stock to other batches of fish on both farms. Samples of RMS affected fish from both farms were returned to our laboratory for further tests, in an effort to learn more about the disease.At this stage, investigations have not shown a clear link between the initial Welsh cases and the English and Scottish cases (where there was clear evidence of disease spread associated with live fish movements), although epidemiological investigations are still ongoing.The affected fish presented with a range of lesions of differing severity. However, despite extensive analysis, including histopathological investigation of preserved material (by light and electron microscopy), mycology, bacteriology and virology, no single potential disease agent could be consistently isolated from affected fish. Histopathologically, the condition was characterised as a non suppurative dermatitis with extensive lymphocytic infiltration. Interestingly, when lesions were examined, it was noted that, even in the very early stages, areas of necrosis and associated inflammatory foci were seen well below the external epithelium in the dermal layer. The first observable stage of the disease is necrosis in the area where the scales are attached to the dermis. Only after this has taken place will scales then be raised or removed and the integument breached, with formation of the characteristic external lesions. Farmers note that lesions are often more obvious after a grading or other stressful handling procedures, which fits with these findings. Another observation was heart pathology in up to 20% of the fish examined (necrosis of cardiac muscle and lymphocytic infiltration).A transmission trial was also run in an effort to see if the disease can be passed to unaffected fish in the laboratory. Twenty RMS affected fish (average size 410.5g) were cohabited with forty naïve rainbow trout (average size 75.4g) in the same tank. Only nine of the naïve fish survived to the end of the ninety-six day trial. Mortalities in the naïve group were not attributable to a particular disease agent and finished by Day 42. Of the remaining nine survivors, four showed external lesions characteristic of Strawberry Disease and RMS (as confirmed by histology). Although these data are interesting, it also serves to illustrate the difficulties of working with a disease where a causative agent has not been identified. There is no evidence at this stage that the mortalities were in any way associated with the syndrome. It is entirely possible that the RMS affected fish were also carrying other diseases that killed the naïve fish. It does appear likely though that the condition was transmitted from the RMS affected fish to the surviving naïve fish. The trial provided other useful information. Out of the eighty RMS affected fish transported to our laboratory, only one died during the course of trial and surviving fish showed signs that the initial lesions had healed up. We also noted no difference between a group of RMS affected fish that were maintained on the diet they had been previously fed on at the farm and a group fed a

SID 5 (2/05) of 27

laboratory control diet. Both groups of fish all appeared to recover from the condition, suggesting a dietary link was unlikely.

03.4.3 FUTURE WORKWe are continuing to investigate outbreaks of RMS as they occur, using a multidisciplinary approach.It is worrying that the disease appears to be so readily transmissible in both the field and laboratory. A number of important questions still remain to be resolved, firstly the nature of the agent causing the disease, and secondly the possible risks to wild populations of salmonids.Agreement on ‘case definitions’ for RMS, Strawberry Disease and other possibly linked conditions to enable accurate differential diagnosis is being sought. This is to enable both the accurate description and the tracking of the spread of the diseases. There is a proposal by Hugh Ferguson from the Institute of Aquaculture to refer to these conditions as Strawberry Disease, with the condition we refer to here as ‘RMS’ termed ‘Coldwater Strawberry Disease’ and what is more commonly understood to be Strawberry Disease in the UK as ‘Warm Water Strawberry Disease’. It is possible that the similar clinical signs for both syndromes are caused by a generalised hyperinflammatory host reaction in the skin layers to the presence of different initial causes (such as different bacterial antigens).

03.5 Other outbreak investigationsCefas Fish Health Inspectorate and other staff undertook a range of diseases investigations during the reporting period and resources supported under FC1151 were used in many cases to help determine whether there was involvement by bacteria or fungi. Two examples of such investigations are reported.

03.5.1 ULCER DISEASE IN COMMON CARP FISHERIES IN THE UK .One such example was investigations of outbreaks of ulcer disease in two common carp fisheries in the UK. Affected fish included mirror carp (Cyprinus carpio) and hybrid carp, which are thought to be a cross between crucian carp (Carassius carassius) and common carp. Managers reported mortality rates over 30% and, although fish were not systematically infected with atypical A. salmonicida, the bacterium was isolated from ulcers.

03.5.2 INVESTIGATION OF A WILD FISH KILL ON THE RIVER TYNE.A wild Atlantic salmon (Salmo salar) and sea trout (Salmo trutta) fish kill occurred on the River Tyne in the summer of 2003. The Environment Agency removed fish on a bi-weekly basis from the affected area and in September six affected fish were removed and returned to the

SID 5 (2/05) of 27

D

Figure 6 a and Figure 6b . Sections of Skin and muscle taken from a RMS lesion Note erosion of the epidermis (arrow head), inflammatory cells in the dermis (D) and into the underlying adipose tissue and musculature, and associated myofibrillar degeneration (arrow)

Weymouth Laboratory for examination. Routine diagnostic testing showed that the affected fish were infected with Vibrio anguillarum (Serotype 01).The water level in the summer and Autumn of 2003 was very low so it is possible that the high water temperatures and crowded, stressful, conditions promoted the onset of vibriosis in the affected fish

Objective 4. Objective as set out in the contract: ‘Establishment of the Bacterial Culture Collection (BCC) and contribution to the Registry of Aquatic Pathology (RAP) Database

Extent to which objective 4 has been met. This objective was fully met over the course of the project.

Details of the methods used and the results obtained, including statistical analysis (where appropriate) and a discussion of the results and their reliability.There are now 2969 isolates listed, almost 200 of them being national collection isolates. 799 isolates have been added since the beginning of 2001. Archival aspects have been addressed by the issue of standard operating procedures (SOP) for the management and maintenance of the collection. This includes controlling how the Access database, on which the collection information is stored, is accessed and managed. All isolates are in long term storage mainly on Protect ®. 150 of those added before 2001 have been re-checked for purity and viability and re-stored as both freeze-dried and Protect® cultures.Amongst other material, samples from L. garviae, C. arthromitus, S. galactiae P. salmonis and Red Mark Syndrome infected fish have been added to the RAP.

05. Objective as set out in the contract: To validate antimicrobial susceptibility tests for all major fish pathogens and to assess the current antibiotic resistance patterns amongst fish pathogens isolated in the UK and that of fish pathogens introduced in the UK via fish imports.’

Extent to which objective 5 has been met. This objective was partially met over the course of the project. The testing methods were fully validated. Less work was done to assess the current antibiotic resistance patterns amongst fish pathogens then was originally planned as a result of resources either being diverted to other projects (in particular the Fish Waste Project F1157, which investigated appropriate methods for inactivating aquaculture pathogens in fish by-products), or to other objectives on this project considered to be of a higher priority by both Defra and project staff (in particular Objectives 2 and 3).

Details of the methods used and the results obtained, including statistical analysis (where appropriate) and a discussion of the results and their reliability.Aquaculture, both freshwater and marine, is affected by a wide range of bacterial diseases which can result in severe economic losses. Many of these diseases can be controlled with vaccines, but protection may be limited and there are some pathogens for which no effective vaccine has been developed.Only a small number of antimicrobial agents are currently approved for use in aquaculture and it is important that these are used as effectively and efficiently as possible.In order to reduce the problem of selection for drug resistance, sensitivity of the pathogen to each of the available drugs should be determined before any treatment commences.Until recently methods for sensitivity testing of bacterial fish pathogens varied widely between laboratories, as did the interpretation of the results produced.

SID 5 (2/05) of 27

A workshop was therefore held at Cefas Weymouth, 24-27 November 1998, with the aim of standardising laboratory methods for the determination of antimicrobial sensitivities of fish pathogenic bacteria. Participants at the workshop included experts from major European and N. American laboratories, with funding by the European Union as a Concerted action by DG XIV under the FAIR programme (FAIR CT 97-3760).After discussion it was agreed that the National Committee for Clinical Laboratory Standards in the US (NCCLS) approach (1997), as illustrated in document M31-T, would be taken as the model for further development. Modified standards for testing antimicrobial sensitivity of aquaculture pathogens have recently been published ( M42-P and M49-P) by the Clinical and Laboratory Standards Institute as (as NCCLS is now known).05.1 Validation of proposed testing guidelinesThis laboratory validated the proposed test methods with respect to A. salmonicida, Y. ruckeri, L. garvieae and V. anguillarum. We used the proposed standards by testing the reference strains suggested, as well as a range of other isolates from each species. In summary, in terms of zone sizes, an acceptable degree of reproducibility was obtained for all drugs tested with Aeromonas salmonicida, V. anguillarum and Y. ruckeri using the method outlined above. Zone sizes obtained for L. garviae were not reliably reproducible from test to test.These methods are now employed as standard operating procedures for testing antimicrobial resistance by CEFAS Weymouth diagnostic function staff.05.2 Patterns of antimicrobial resistance in Y. ruckeri. In all 27 strains of Y. ruckeri were tested for antimicrobial resistance to 10- antibiotics by the developed plate assay method. Results indicated that there were little differences in patterns of antimicrobial resistance between the different isolates.

Main Implications of the FindingsThe main implications of this study are that diseases caused by bacterial and fungal agents remain a highly significant, possibly increasing, threat to farmed and wild fish populations in England and Wales. The general range of conditions investigated under FC1151 illustrates how threats can come from both agents that have evolved to circumvent existing control strategies, as well as novel diseases. Yersinia ruckeri, the agent responsible for Enteric Redmouth, is an example of an endemic pathogen shown here to have altered in its properties to evade existing vaccination-based control strategies. Red Mark Syndrome, by contrast, is an example of a disease of farmed rainbow trout that has emerged in the last two years.It is possible that some conditions may become more of a threat in the future to both farmed and wild populations. Under this project, effort was put towards assessing the likely threat to native UK fish species from Lactococcus garvieae and other Gram positive organisms. It is concluded that these organisms are likely to pose a significant threat in the future. Grayling were shown to be highly susceptible with coarse fish species demonstrated to be moderately susceptible and also to potentially be able to carry the disease. As there is some evidence that these diseases can be of increased risk at higher water temperatures, the specific threat from Lactococcosis, and a range of other diseases, may well increase as a consequence of temperature changes associated with future climate change. We are now in a position to successfully discriminate Gram positive pathogens rapidly as a result of work done under this project.Another major implication of the findings is that emerging bacterial and fungal diseases, as with other diseases, are best investigated using a flexible, multidisciplinary approach, as practiced here. It is vital that there is a good link between those in close contact with potentially infected fish populations (farmers, river bailiffs, veterinarians, Fish Health inspectors etc.) and a wide range of experts from different disciplines, who are able to respond quickly to assess the threats as they are reported. Such flexible contracts do not always fit well within the traditional research proposal framework, with well-defined objectives and attendant milestones decided at the beginning. However to provide Defra with the timely authoritative advice it requires, such an approach is necessary.

SID 5 (2/05) of 27

Possible Future Work Emerging bacterial and fungal diseases should continue to be investigated on Defra’s behalf using a flexible, multidisciplinary approach. It is suggested that this be done within the overall framework of Project FC 1166 ‘Characterisation and Pathogenesis of fish and shellfish emerging diseases’. Of the disease threats investigated or identified under this project further work should be done to determine:

1. The mechanisms by which Yersinia ruckeri has evolved to evade existing control strategies

2. Epidemiology and aetiology of rainbow trout gastroenteritis with a view to developing methods for its diagnosis and control.

3. Epidemiology and aetiology of Red Mark Syndrome with a view to developing methods for its diagnosis and control.

References cited Ahne W., Popp W. and Hoffman R. (1982). Pseudomonas fluorescence as a pathogen for fish. Bull. Eur. Ass. Fish Path. 2: 56-57. Algoet M. (2001) Investigations into the causes of The Kuwait Fish Kill – Summer 2001. Internal CEFAS reportAmey, S.D. (2005) The role of bacteria associated with seahorses (Hippocampus sp.): on their health and survival. MRes thesis. University of Plymouth 110 pp.Austin B. and D.A. Austin. 1999. Bacterial fish pathogens: Diseases of farmed and wild fish. Springer Praxis. 457pp.CLSI (2005) Methods for antimicrobial disk susceptibility testing of bacteria isolated from aquatic animals: proposed guideline. CLSI document M42-P (ISBN 1-56238-577-1)CLSI (2005) Methods for broth dilution susceptibility testing of bacteria isolated from aquatic animals: proposed guideline. CLSI document M49-P (ISBN 1-56238-576-3)Cowan and Steel’s Manual for the Identification of Medical Bacteria. 3rd Edn.: Cambridge University Press 331pp.Dierckens K.R., Vandenberghe J., Beladjal L., Huys G., Mertens J. and Swings J. (1998). Aeromonas hydrophila causes ‘black disease’ in fairy shrimps (Anostraca; Crustacea) J. Fish Dis. 21: 113-119.Gaultier C. (2006) Identification phenotypique des bacteries ichtyopathogenes du genre Vibrio. These pour le Diplome d’Etat de Docteur Veterinaire. Ecole Nationale Veterinaire de Nantes. Pending publicationGhittino C., Prearo C., Ghittino M. and Eldar A. (1998). Recent knowledge on warm water ‘streptococcoses’ in rainbow trout. Boll. Soc. Ital. Patol. Ittica. 10(23): 43-50.Hiney M.P., Smith P.R. and Bernoth E.M. (1997). Covert Aeromonas salmonicida infections, pp 54-97 in: Bernoth E.M., Ellis A.E., Midtlyng P.J., Olivier G. and Smith P., eds. Furunculosis: Multidisciplinary fish disease research. Academic press. San Diego, California. 529pp. Kozinska, A. and Peokala, A. (2004) First isolation of Shewanella putrefaciens from freshwater fish- a potential new pathogen of fish. Bull. Eur. Ass. Fish Pathol. 24(4) 189-193.Maslow J. and Mulligan M.E. (1996). Epidemiologic typing systems. Infect. Control Hosp. Epidemiol. 17:595-604.Muzquiz J.L., Royo F., Ortega C., de Blas I., Ruiz I. and Alonso J.L. (1999). Pathogenicity of streptococcosis in rainbow trout (Onchorhynchus mykiss): dependence on age of diseased fish. Bull. Eur. Ass. Fish Path. 19(3): 114-118.NCCLS (1997): M31T Performance standards for antimicrobial disk and dilution susceptibility tests for bacteria isolated from animals; tentative standard. ISBN 1-56238-320-2Nedoluha, P. C. and Westhoff, D. (1997). Microbiology of striped bass grown in three

SID 5 (2/05) of 27

aquaculture systems. Food Microbiology 14: 255-264.Olive, D.M. and Bean, P. (1999) Principles and Applications of Methods for DNA-Based Typing of Microbial Organisms. J. Clin. Microbiol.37, 1661-1669Pereira F., Ravelo C., Toranzo A.E, Romalde A.J. (2004). Lactococcus garvieae, an emerging pathogen for the Portugese trout culture. Bull. Eur. Ass. Fish Path. 24(6): 274-279.Ravelo C., Magarinos B., Romalde J.L. and Toranzo A.E. (2001). Conventional versus miniturized systems for the phenotypic characterization of Lactococcus garvieae strains. Bull. Eur. Ass. Fish Pathol. 21(4) 136-144.Roald, S.O. and Hastein, T. (1980). Infection with an Acinobacter-like bacterium in Atlantic salmon. pp. 154-156 In: Ahne, W. (Ed) Proceedings 3rd COPRAQ Conference, Munich, Oct. 1979.Streulens MJ, (1998). Molecular epidemiologic typing systems of bacterial pathogens: current issues and perspectives. Mem. Inst. Oswaldo. Cruz., Rio de Janeiro, 93:581-585. Tenover FC, Arbeit RD, Goering RV, the Molecular Typing Working Group of the Society for Healthcare Epidemiology of America (1997). How to select and interpret molecular typing methods for epidemiological studies of bacterial infections: a review for healthcare epidemiologists. Infect. Control Hosp. Epidemiolo., 18:426-439Toranzo, A.E., Barja, A.M., Romalde, J.L. and Hetrick F.M. (1989) Association of Aeromonas sobria with mortalities of adult gizzard shad, Dorosoma cepedianum Lesuer. J. Fish Dis. 12: 439-448.Udaci, M. C. , Reganuult, B. and Grimont, P. A. D. (2001) Identification by in situ hybridisation of segmented filamentous bacteria in the intestine of diarrheic trout (Oncorhynchus mykiss). Res. Microbiol. 152, 67-73. Verner – Jeffreys D. W., Shields, R. J., Bricknell, I.R., Birkbeck, T. H. (2003) Changes in the gut-associated microfloras during the development of Atlantic halibut (Hippoglossus hippoglossus L.) larvae in British hatcheries. Aquaculture 219, 21-42.Zlotkin A., Hershko H. and Eldar A. (1998). Possible transmission of Streptococcus iniae from wild fish to cultured marine fish. Appl. Environ. Microbiol. 64 (10) 4065-7.

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.

SID 5 (2/05) of 27

Presentations and publications arising from this project

Algoet, M., Driscoll, J., Bayley, A. and Roberts, E.(2003) Evaluation of Biolog's microlog system for the identification of gram positive cocci pathogenic for fish. Poster presented at 11th International Conference on Fish and Shellfish Pathology, September 2003, Malta

Algoet, M. Bayley, A. and Roberts, E. (2005) Susceptibility of selected freshwater course and salmonid fish species to Lactococcus garvieae Poster presented at 12th International Conference on Fish and Shellfish Pathology, September 2005, Copenhagen, Denmark.

Amey, S.D. (2005) The role of bacteria associated with seahorses (Hippocampus sp.): on their health and survival. MRes thesis. University of Plymouth 110 pp.Mackie M., Bayley A. and Algoet M. (2003) Investigations into the causes of a new severe chronic skin condition affecting Atlantic salmon pre-smolts. Poster presented at 11th International Conference on Fish and Shellfish Pathology, hold from 11-16 September 2003, Malta

St-Hilaire, S., Mander, B., Bayley, A. and Gardiner, R. (2005) Ulcer disease in common carp fisheries in the UK. Fish Veterinary Journal 8, 101-106

St-Hilaire, S., Gubbins, M. and Bayley, A. (2005) Investigation of a wild fish kill on the River Tyne . Fish Veterinary Journal 8, 107-113

Verner – Jeffreys, D. W., Nakamura, I, and Shields, R.J (2006) Egg-associated microflora of Pacific threadfin, Polydactylus sexfilis and amberjack, Seriola rivoliana, eggs. Characterisation and properties. Aquaculture 253, 184-196

Verner – Jeffreys, D.*, Algoet, M., Peeler, E., Feist, S. and Bateman, K. (2006) Studies on Red Mark Syndrome. Finfish News (In press) http://www.cefas.co.uk/Publications/

Verner-Jeffreys, D. and Algoët, M.. 2005. Fish medicines and feed additives: a guide to the regulatory implications of their use in UK aquaculture. Trout News 39: 19-22.

Papers in preparation (to be submitted by July 2006)Algoet M., Bayley A. and Roberts E. Susceptibility of selected UK freshwater coarse and salmonid fish species to Lactococcus garvieae. To be submitted to be Diseases of Aquatic Organisms.

Algoet M., Driscoll J., Bayley A. and Roberts E. Evaluation of Biolog’s Microlog system for the identification of Gram positive cocci pathogenic for fish to be submitted to Bulletin of the European Association of Fish Pathologists.

Algoet M., Mackie M. and Roberts E. Investigations into the causes of a new severe chronic skin condition affecting Atlantic salmon pre-smolts. Paper to be submitted to Bulletin of the European Association of Fish Pathologists.

Gaultier C. (2006) Identification phenotypique des bacteries ichtyopathogenes du genre Vibrio. These pour le Diplome d’Etat de Docteur Veterinaire. Ecole Nationale Veterinaire de Nantes. Pending publication

Meetings attended:Glasgow Aquaculture in Spring 2004British Marine Finfish Association meeting in Oban in Autumn 2004VIth EFARO technical workshop – Diversification in Aquaculture in Feb 05Informal meetings with representatives of the farming industry and fish health professionals11th International Conferences on Fish and Shellfish Pathology , September 2003, Malta 12th International Conferences on Fish and Shellfish Pathology September 2005, Copenhagen

SID 5 (2/05) of 27

http://www.cefas.co.uk/Publications/

SID 5 (2/05) of 27

general enquiries on this form should be made...

Documents