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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.

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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 FC1137

2. Project title

Characterisation and pathogenesis of parasites and diseases in fish and shellfish stocks.

3. Contractororganisation(s)

CEFAS Weymouth Laboratory,Barrack Road,The Nothe,Weymouth,Dorset. DT4 8UB     

54. Total Defra project costs £ 361084

5. Project: start date................ 01 April 2000

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 main aim of the project was to facilitate the provision of authoritative scientific advice to Defra on the significance of a wide variety of pathogens or potential pathogens of fish and shellfish. The objectives were as follows:

To obtain detailed information on the pathogenicity and identity of a wide variety of parasites and other pathogens affecting fish and shellfish stocks.

To identify those pathogens detected in exotic fish species that may pose a threat to native stocks.

To improve knowledge of the normal structure, histology and parasite fauna of marine fish species of potential for aquaculture.

To establish the Registry of Aquatic Pathology (RAP) as an internationally recognised resource and depository for pathological specimens and parasites.

This project has provided a wealth of information on the health status of wild fish and shellfish in UK waters. The most important finding from the studies conducted under this project to date is that infections with parasites in juvenile coarse fish can have a significant detrimental effect at the population level. This research has highlighted the strong probability that chronic disease is an important factor affecting year class strength in wild fish and has significant implications for stock assessments and modelling of freshwater and marine fisheries. The majority of disease conditions reported were caused by protistan and metazoan parasites. The effect of acute diseases in wild fish caused by viral and bacterial pathogens is less certain. A number of parasites new to science have been described, including gyrodactylid species, in collaboration with Dr Shinn at the Institute of Aquaculture, University of Stirling, as part of development of an intelligent system for the rapid identification of these parasites. Surveys on the parasite fauna of wild salmonids and grayling have revealed the presence of previously unrecognised parasites in the UK and extended the known host range for others such as a coccidian infecting the intestine of brown trout and grayling. Investigations into the parasites and other pathogens of non-native fish identified a number of parasites. Of particular note was the discovery, in collaboration with CEH, of the role of topmouth gudgeon as a healthy host of Sphaerothecum destruens (rosette agent) in the transmission of this pathogen to another non-native species, the sunbleak. Further studies are required to determine the threat of this disease on native fish species. Studies on scallops have confirmed previous Defra funded work that

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several fisheries are affected by disease, but at low levels with little pathology severe enough to cause large scale mortalities. Cultured stocks appear to harbour more pathogens. Other shellfish such as edible crab have been investigated following reports of diseased animals in catches. A highly significant pathogen (Hematodinium) closely related to one known from Norway lobster was confirmed as the cause of the infections. The disease was named ‘Pink crab disease’ in a publication describing the condition. Viral diseases affecting brown shrimp at high prevalences and infections with yeasts and a Paramarteilia -like species were found in edible crabs. Effects at the population level are yet to be determined. Examples of much of the material examined have been deposited in the RAP, which now constitutes a significant resource used by CEFAS staff and visiting scientists. Its value continues to grow as more specimens are deposited. Numerous presentations and scientific publications, including book chapters (BSAVA Manual of Ornamental Fish, and ‘Phylum Myxozoa’ in Fish Diseases and Disorders ed P. T. K. Woo) have emanated from this work.

This project provided scientific advice on the significance of parasites and other pathogens detected in wild and cultured fish and shellfish stocks from the UK by undertaking screening of selected freshwater and marine species of ecological importance or of current and potential use in aquaculture. The project was designed to complement the responsibilities of the Fish Health Inspectorate (FHI) by undertaking detailed assessments of the pathological impact of pathogens in cases of disease outbreaks as well as assessing the significance of parasites recovered from imports of ornamental fish.

The information obtained provides essential baseline data on normal structure of healthy animals and possible disease threats to those species in particular and others in inland and coastal waters. Data obtained contributes to Parliamentary Questions and enquiries from the public pertaining to 'health' of marine fish. Investigations undertaken under this project have provided valuable insights into the significance of diseases affecting wild fish and shellfish stocks. In particular, that disease can be a highly significant factor influencing population success and existing wild fish population models should incorporate the disease element.

There is a continuing need to be aware of the range of pathogens exerting an adverse influence on fish and shellfish species in the UK aquatic environment. Particularly those which may be detrimental for recruitment to adult populations and year class strength (i.e. affecting stock size and survivability). Our ability to accurately diagnose previously unrecognised pathogens is dependant on familiarity with a wide range of pathogens or potential pathogens infecting hosts from a variety of sources within the EU and further afield. The RAP is extremely valuable for assisting in these diagnoses and for training purposes and its value increases with time. Following identification, it is essential to determine pathogenicity using histopathology and possibly transmission studies. Long-term data sets on parasite disease incidence are needed in order to assess population effects. For this, studies such as those conducted under F1137 will continue to be required.

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 1. To obtain detailed information on the pathogenicity and identity of a wide variety of parasites and other pathogens affecting fish and shellfish stocks.

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Samples of fish and shellfish were obtained from a variety of sources. Parasite surveys of wild salmonids were undertaken using material from investigations carried out by the Fish Health Inspectorate (FHI) and the Environment Agency (EA). Specimens of juvenile and adult coarse fish were also supplied by collaboration with the EA. Shellfish, including scallops and various crustacean species were obtained from a variety of sources, either as a by-catch on board RV’s Cirolana and CEFAS Endeavour during annual environmental monitoring activities (contract AE003), from material examining biological effects of contaminants in estuarine organisms (contract CDEP 84/5/312) or from sites in the environs of Weymouth and Portland. Material from non-native fish species originated from the FHI and collaborative studies with the Centre of Ecology and Hydrology (CEH Winfrith).

The principle laboratory methods used to obtain information on the pathogenicity of parasites and other pathogens were histological analysis and parasitological techniques. Samples for histopathology were preserved either in the field or on receipt at CEFAS Weymouth Laboratory in neutral buffered formalin (NBF) or ethanol. Fixed tissues were processed using conventional protocols in a vacuum infiltration processor, embedded in wax, trimmed, sectioned at 5 microns thickness and placed on glass slides. Sections were routinely stained with haematoxylin and eosin (H&E) and examined with a photomicroscope at appropriate magnifications. Additional specific stains for tissue elements and micro-organisms were applied as required. Electron microscopy was undertaken on selected material in order to characterise fine structure of pathogens and to describe cellular interactions.

Determination of parasite infections was also undertaken using a combination of light microscopy techniques such as phase and interference contrast microscopy to elucidate morphological characters of taxonomic importance. Ultrastructural examination of fixed material was used to determine fine structure of features used to discriminate species. In particular, this approach was used for monogenean parasites of fish. Selected material from these studies was deposited in the Registry of Aquatic Pathology (RAP) (see below).

Freshwater fish investigations

Detailed examination of histological material from wild salmonids and coarse fish for the presence of diseases caused by parasites and microbial pathogens was undertaken. Pathological changes which may be related to environmental contaminants including endocrine disrupters were also recorded.

Examination of material obtained from a salmonid survey undertaken by the FHI involving 47 sites across England and Wales, identified a high prevalence of intestinal coccidiosis caused by a protozoan parasite (tentatively identified as Eimeria truttae) in brown trout and Atlantic salmon. Brown trout (n=132) taken from 7 of 9 sites, harboured the infection at prevalences between 13.33% from the R. Culd to 100% from the River Esk (9 fish examined). Salmon (n=740) from 22 of 32 sites harboured the infection at prevalences between 3.33% from the River Coquet in Northumberland and 86.2% from the River Ceiriog in SW Wales. Of the 36 sea trout obtained from 6 sites, only a single fish from the R. Avon was found to be infected. A single grayling from the Tees also harboured the parasite. The infection has not been previously recorded in Atlantic salmon or grayling. Significant host response was only apparent in heavy infections. Renal infections with Chloromyxum sp. were identified at high prevalences in salmon and brown trout from most sites with minimal host response recorded. Gill epitheliocystis in salmon caused by a rickettsia-like agent was detected at three sites (Rivers Caldew and Lune and Clwedog Dam). Although prevalence was 50% at Clwedog Dam the intensity of infection was not considered severe enough to cause mortality.

Two cases of intersex in salmon from the rivers Ceiriog and Esk (SW Wales and Northumbria respectively) were detected, these being the first cases of this condition reported in salmon from the UK. A further case was found affecting a 3-spined stickleback. In addition, a case of liver disease was detected in a brown trout from the River Tyne. The cause could not be determined but the histological features were similar to those reported after exposure to certain algal biotoxins. Further samples are required to determine whether the condition is more widespread or is affecting other fish species.

Grayling were collected from the River Itchen near Southampton in December 2002 and examined for parasites and histopathology. All fish were infected by the digenean Crepidostomum farionis wth a maximum of 145 individual parasites in one fish. The mean number of C. farionis in the sample was 54. Additionally, grayling from the Itchen were infected with the digeneans C. metoecus and Allocreadium tranversale; the nematodes Cystidicoloides ephereidarum, Cytidicola farionis, Spinitectus gordoni and Truttaedacnitis truttae, and two acanthocephalan species Echinorhynchus truttae and Neoechinorhynchus rutili. Of most interest was the finding of Spinitectus gordoni in one grayling this being a new record for the

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UK and for this host. The nematode has previously only been recorded in brown trout and salmon in northern Spain. Gyrodactylids (G. thymalli) were sampled from these fish for analysis using shape recognition software developed by Dr Shinn (University of Stirling). Histopathological examination of the organs of these grayling revealed few significant pathologies with the exception of gill aneurysms in several fish. Host response to the parasites identified was minimal.

Information obtained previously on an epizootic of Dermocystidium in bullheads (Cottus gobio) was prepared for publication during the period of this contract. Bullheads with macroscopic external cysts on the skin and fins measuring up to 3 mm in diameter were detected in the River Allen and its tributaries in southern England by EA staff. The prevalence of these cysts was up to 50% at some sites. Examination of cyst contents revealed the presence of numerous spores, typical of the genus Dermocystidium, measuring 8µm in diameter. The parasite developed within well-defined cysts, which were located in the hypodermal connective tissues of the host. No cysts were present on the fins of any of the fish examined. Histological examination of these revealed a cyst wall consisting of an inner layer of dense eosinophilic material similar to that reported for Dermocystidium spp. forming coenocytic hyphae. No evidence was found of systemic infection or hyphal formation. Spores contained a prominent refractile body, which gave a weakly positive reaction for polysaccharides with the periodic-acid Schiff reaction (PAS), and was positively stained with acidic dyes. Several examples of ruptured cysts were seen in histological sections and in some of these cases, the host epithelial layer was breached allowing release of the spores to the environment. Morphological features of, and host response towards the Dermocystidium sp. in bullheads were compared to similar infections in salmonids and other freshwater fish species (Feist et al., 2004).

Studies in collaboration with Dr Andrew Shinn (University of Stirling) were conducted to examine the Gyrodactylus fauna of freshwater fish. The survey of freshwater fish further confirmed the absence of G. salaris in UK waters, in particular on non-salmonid hosts. The studies provided further examples of Gyrodactylus spp. for inclusion in a neural network program for the identification and discrimination of G. salaris developed by Dr Shinn for use by CEFAS and others as a diagnostic tool. Material from this work will be included in a definitive key and synopsis of UK Gyrodactylus spp. currently in preparation. Twenty species of native freshwater fish were examined for the presence of Gyrodactylus spp. Of these, 11 fish species harboured Gyrodactylus spp. Brown trout (Salmo trutta) were infected with G. derjavini and G. truttae, Atlantic salmon (Salmo salar) were infected with G. derjavini, rainbow trout (Onchorhynchus mykiss) were infected with G. derjavini and G. truttae and grayling (Thymallus thymallus) were infected with G. thymalli. Minnow (Phoxinus phoxinus) contained the most number of species, being infected with G. pannonicus, G. aphyae, G. limneus, G. phoxini, G. magnificus and G. macronychus. Stone loach (Barbatula barbatula) were infected with G. tonii, bullheads (Cottus gobio) were infected with G. rogatensis, bream (Abramis brama) were infected with G. elegans, carp (Cyprinus carpio) were infected with G. katharineri and G. cyprini, tench (Tinca tinca) were infected with G. derjavini, G. tincae and G. cyprini and dace (Leuciscus leuciscus) were found to harbour what appears to be a currently undescribed Gyrodactylus sp.

In addition to the freshwater species, a limited number of marine fish were examined for the presence of gyrodactylids. These samples were used to confirm the specificity of the neural network system, although the data was not incorporated into the statistical classifier. During the summer of 2003 marine fish, including gadoids, gobies, weevers, dragonets, argentines, gurnards, pogges, bull rout and blennies were collected in the English Channel and the Irish Sea on the RV CEFAS Endeavour. Small fish were fixed whole and for larger fish, gills were removed and fixed in 10% NBF or ethanol. On return to CEFAS Weymouth laboratory, the fish were screened for monogenean infections using a stereomicroscope. The NBF-fixed fish were rinsed in distilled water which was passed through a 50-micron pore size filter before being transferred to distilled water in a petri dish, where the gills, fins and washings were examined. Monogenean specimens were removed with a Gilson pipette or paintbrush. Gyrodactylid specimens were air-dried onto glass slides. Specimens were treated on the slide using 2.5μl proteinase-k based digest and 1.5 μl stop solution (1 part formaldehyde to 1 part 100% glycerine) and cover-slipped, using clear nail varnish as a sealant. The parasites were studied using a Nikon E800 microscope. Representative images were captured and later measured using a video camera mounted to the microscope and linked to a LUCIA Screen Measurement image analysis system (Nikon UK Ltd).

Of 21 species of fish examined, Gyrodactylus spp. were only recorded on dab (Limanda limanda), herring (Clupea harengus), poor cod (Trisopterus minutes), two-spotted goby (Gobiusculus flavescens), sand goby (Pomatoschistus minitus), and painted goby (P. pictus). Eleven previously unrecorded Gyrodactylus spp. were found. Members of this genus appear to have been overlooked in many surveys of marine fish parasites, possibly due to their small size. It is likely that there are many more species yet to be described in marine fish worldwide. Advances in the knowledge of gyrodactylid taxonomy can be gained from

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examining the fauna of marine hosts and such studies offer potential for greater understanding of parasite biodiversity of marine fish species. Whilst the species of fish examined are highly unlikely to be used as aquaculture species, their parasite fauna remains a potential threat to the successful culture of other marine species due to the possible transfer of parasites from wild fish to farmed stocks. A baseline knowledge of the gyrodactylid fauna of native socks would assist in the rapid identification of any parasites that may become problematic in the future.

Studies on population level effects of disease

Materials and methods

For studies to examine the role of disease on population success, a total of 1053 young of the year (0+) cyprinid fry, comprising 563 chub (Leuciscus cephalus) and 490 roach (Rutilus rutilus), were collected semi-quantitatively from Acaster, Beningbrough, and upstream of Linton Weir on the river Ouse, Thornton Bridge on the River Swale, and from Boston Spa on the river Wharfe in Yorkshire, northern England every September between 1993 and 2002. These fish were a sub-sample of approximately 4600 fish collected at 28 sites on 15 rivers, comprising 1000 roach, 1300 chub, 500 dace (Leuciscus leuciscus) and 1800 minnow (Phoxinus phoxinus). Fish were caught using three sweeps of a 20m × 2m micromesh seine net set along the river margin at each site, killed by over anaesthetising in MS222 and fixed in 10% neutral buffered formalin (NBF). On return to the laboratory, fish were identified to species, fork length was taken and the fish stored in NBF by site and species. For histological studies a sub-sample of fry of each species from each site for each year were taken. A maximum of 30 fish of each species per site were examined. Numbers of myxozoan cysts and individual digeneans in the musculature were enumerated and the location of, and pathological changes associated with, all infections were noted.

Measures of parasite infection used were prevalence and abundance. For myxozoan cysts in the musculature, variance to mean ratios (V:M) were calculated and the power exponent for Taylor’s power law was calculated from linear regressions of Log variance versus Log mean values. Exponents were determined from the slope of the regression line. Year class strength (YCS) data was only available for roach and chub from the rivers Wharfe and Ouse and was calculated with data provided by the Environment Agency. In addition daily water temperature between 1993 and 1999 in the river Ouse were used to calculate the cumulative degree-days greater than 12°C between April and September inclusive for each year. Daily river discharge data for the river Ouse was obtained from the Environment Agency multipath ultrasonic flow gauging station at Skelton and used to calculate the annual cumulative number of discharge days above the basal discharge rate between April and September inclusive. Position of the north wall of the Gulf Stream (NWGS) between 1993 and 2000 was obtained from the Plymouth Marine Laboratory.

Following determination of correlations between variables and responses, linear and multiple regression models were produced using MINITAB (ver. 13.20) and confirmed using stepwise regressions.

Results

There was considerable spatio-temporal and interspecific variation in parasitism throughout the study. In general terms, chub were the most parasitised in the number of parasite species, prevalence and abundance and pathological responses were greatest in this species. Roach have fewer parasite species compared with chub, show a lesser pathological response and parasites are found at lower prevalence and abundance. Despite the wide variations noted, patterns of parasite prevalence and abundance was similar across all sites with fish collected in 1993, 1997 and 1998 having comparatively higher levels of parasitism compared with 1994, 1995 and 1996. Parasite prevalence and abundance was lowest in the 1995 year class for all species examined. For myxozoans in the muscle, abundance of cysts followed a similar pattern to that seen in the prevalence data. Around forty different parasite species were identified in the study and with the exception of Rickettsia-like organisms (RLO’s), no bacterial or viral induced pathology were noted in the fish examined. Typically, the parasites found had direct lifecycles or for those with a complex lifecycle, via water borne transmission stages, typified by the digenean Bucephalus polymorphus and by myxozoans.

Year class strength (YCS) in chub and roach was generally lowest in the 1993 and 1998 cohorts and highest in the 1994 and 1995 year classes.

The best model to describe the impact of disease on length of chub was from fish collected at

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Beningbrough (Length chub = 34.9 + 0.0771 (Myxobolus spp.) – 1.38 (Myxobolus spp. abundance) P = 0.002, R2 = 100) which showed that length was negatively correlated with the prevalence and abundance of Myxobolus spp. in the muscle, even with the inclusion of environmental data. Linear models further supported this assertion for the Ouse at Beningbrough (Length chub = 36.2 – 0.147 (Myxobolus spp.) P= 0.063, R2 = 62.1 and Length chub = 33.2 – 2.33 (Myxobolus spp. abundance) P = 0.029, R2 = 94.2%)

Chub length was also positively correlated with the presence of the digeneans B. polymorphus and Phyllodistomum sp. (Length chub = 8.02 +1.10 (Phyllodistomum sp.) + 0.199 (Bucephalus polymorphus), P = 0.014 R2 = 94.2%; Length chub = 28.1 +1.00 (Bucephalus polymorphus) – 0.268 (Myxobolus spp. abundance), P = 0.029 R2 = 97.1%) and Myxobolus buckei in the spinal column at different sites. Fitting environmental variables did not significantly increase the explained variation (R2) with the exception of chub length at Thornton Bridge, which in addition to M. buckei, included the position of the NWGS in January (Length chub = 23.4 + 0.926 (NWGS January) + 0.766 (Myxobolus buckei) P = 0.049 R2 = 86.5%).

The best model to describe the effect of disease on chub relative abundance was for chub caught in Beningbrough and included the prevalence of Myxobolus spp. in the musculature and position of the NWGS in July (Relative abundance chub = 58.1 – 0.0371 (Myxobolus spp.) – 18.0 (NWGS July) P = 0.069, R2

= 99.5%). At the different sites, chub relative abundance was also correlated with the digenean Phyllodistomum sp. (Relative abundance chub = 58.7 – 5.09 (Phyllodistomum sp.) P = 0.005, R2 = 95%; equation 9: P = 0.036, R2 = 96.4%), Myxobolus spp. in the musculature (Relative abundance chub = 64.6 – 0.088 (Myxobolus spp.) – 5.52 (Phyllodistomum sp.) P = 0.036, R2 = 96.4%, Relative abundance chub = 28.5 – 6.15 (Myxobolus spp. abundance) + 10.1 (ciliates) P = 0.03, R2 = 97%) M. buckei (Relative abundance chub = 17.3 + 0.775 (Myxobolus buckei) P = 0.055, R2 = 75.7%) and M. macrocapsularis (Relative abundance chub = 22.2 + 4.02 (Myxobolus macrocapsularis) P = 0.075, R² = 58.8%).

A significant relationship between YCS and the prevalence of RLO’s and between YCS and the variance to mean ratio of muscle myxozoans was found in chub from the river Ouse (YCS chub = 217.0 – 2.18 (RLO’s) P = 0.006 R2 = 88.0%; YCS chub = 245.0 – 12.9 (variance to mean ratio of Myxobolus spp.) P = 0.016 R2 = 88.9%). However, a positive relationship was noted in YCS of chub from the river Wharfe with B. polymorphus (YCS chub = 31.0 + 17.3 (Bucephalus polymorphus) P = 0.001 R2 = 98.3%). A complex model including environmental data and other pathogens showed a strong correletaion for the impact on YCS (YCSchub = 256 + 26.2 (RLO’s) - 1.00 (Myxobolus spp. in muscle) + 13.7 (M. macrocapsularis) - 2.53 (Bucephalus polymorphus) P = 0.001 R2 = 100%)

Fewer models were developed to demonstrate a link between disease and roach compared with chub and were related to the presence of B. polymorphus in the musculature, Trichodina in the gills, myxozoan plasmodia in the kidney tubule epithelium and to a lesser extent, Coccidia in the intestinal epithelium and RLO’s in the gills. Relative abundance of roach was positively correlated with disease (Relative abundance roach = 12.9 + 9.32 (Bucephalus polymorphus) P = 0.011 R2 = 83.2%; Relative abundance roach = 39.3 + 8.75 (myxozoan plasmodia in kidney) P = 0.038 R2 = 80.8%; Relative abundance roach = 9.86 + 0.368 (Bucephalus polymorphus) + 10.7 (RLO’s) P = 0.027 R2 = 76.5%; Relative abundance roach = 57.8 + 6.94 (myxozoan plasmodia in kidney) – 3.86 (Coccidia) P = 0.028 R2 = 97.2%). As with chub, YCS was positively correlated with the presence of B. polymorphus (YCS roach = 51.0 + 22.4 (Bucephalus polymorphus) P = 0.004 R2 = 80.2%), but unlike chub, YCS was negatively correlated with the presence of Trichodina spp. in the gills (YCS roach = 103 – 4.10 (Trichodina spp.) P = 0.003 R2 = 99.3%; YCS roach = 127 – 4.64 (Trichodina spp.) – 0.031 (degree days) P = 0.011 R2 = 100%).

Discussion

The current study has, for the first time, demonstrated a correlation between disease levels in juvenile fish and their role in determining year class strength and recruitment success. Whilst it has been suggested from previous studies that disease may impact on population success, these studies have tended to be conducted post-mortality and once population structure has been fixed by a series of abiotic and biotic factors, including disease. Previous studies by the Environment Agency and University of Hull on the same population of fish in the rivers Ouse, Wharfe and Swale suggested that the main drivers for population success were the position of the north wall of the Gulf Stream (NWGS), temperature and flow rate. It was considered by those authors that the position of the NWGS determined the summer temperature of the rivers, which in turn enhanced fish growth at higher temperatures. Those authors failed to consider the role that parasites and diseases may have on fish survivability.

Due to the nature of the infection process, the juvenile fish examined in the current study fish are unlikely to be able to avoid the infectious stages released by the invertebrate hosts and transmission of these

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infectious stages may be exacerbated in the shallow, warm river margins in which the juvenile fish reside in the first few months of life. It is during the period within the margins that juvenile fish attempt to attain a maximum size prior to moving into the main body of the river. Newly hatched larvae are poor swimmers due to the limited muscle structure and small size. As the fish increase in length, their ability to maintain station in high water flows increases due to an increase in muscle mass. Episodic flooding events either during the summer months, or later in autumn and winter may cause direct fish mortality by washing the smaller fish out of the riparian zones. Therefore, factors that alter the growth rate and length of the fish will impact on host survival. During the current study, models were developed that demonstrated a correlation between parasitism and fish length for chub.

A strong negative correlation between Myxobolus spp. abundance in the muscle and chub length was determined. There are multiple effects of muscle infections on the fish host. Myxozoan cysts in individual muscle fibres lead ultimately to the destruction of the infected muscle fibre and following the host response, adjacent muscle fibres may be destroyed. Loss of muscle function in these hosts will restrict the fishes ability to actively swim to catch prey food items, thus a reduction in energy intake would be expected. This would then be translated into reduced growth and a smaller overall size. Consequently, the smaller sized would be expected to be less able to maintain a suitable position in the water column during episodic flooding events, and more susceptible to predator induced mortality as their ability to swim away from predators would be reduced.

Conversely, a positive relationship was found between chub length and the prevalence of Bucephalus polymorphus in the musculature of chub. This may be explained by the fact that, unlike myxozoan infections which predominately occur in the first few weeks of life, infectious cercarial stages may be present over a protracted number of months in these rivers. The presence of metacercaria in different host organs and at different developmental stages would support this view. Larger fish in the riparian zone would therefore be expected to have a higher abundance and prevalence of digeneans in the musculature compared with smaller fish, which may have hatched at a later date. Alternatively, the positive relationship between length and B. polymorphus in the muscle may be due to the spawning behaviour of adult chub which are known to possess the ability to spawn more than once in the summer. Larger juveniles in the population may represent an early batch or batches of fish that hatch out during the period when digenean cercaria are present and that the smaller, less infected fish are those that hatch later in the season, outwith the critical main period for infection. Due to the nature of sample collection and analysis, it was not possible during the current study to determine which process was operating in the chub populations and could be addressed by assessing the levels of parasitism in monthly samples of chub throughout the summer months post-hatching. It follows that the positive correlation between the presence of B. polymorphus and roach YCS may be due to the larger individuals which hatch earlier are able to survive flooding events thus contributing to a strong year class.

There was a strong negative correlation between chub YCS in the River Ouse and RLO’s and Myxobolus spp. infections in the musculature. As described above, the impact of myxozoan infections in the muscle are likely to disrupt the normal function and architecture of the skeletal muscle and impact on the host’s ability to capture prey items, maintain position in high water flows and to avoid predators. Host losses associated with these factors would be expected to be reflected in the YCS, as demonstrated here. Chub YCS was also strongly correlated with the variance to mean ratio of Myxobolus spp. in the muscle. Variance to mean ratios can be a measure of the degree of overdispersion within a parasite population, with higher values suggestive of a greater degree of overdispersion, whereby most hosts have low levels of infection with a small proportion of the host population being heavily parasitised. The more heavily infected individuals would be killed by the heavy parasite burdens thus allowing transmission of the parasite to the next host as appropriate and continuing the lifecycle. However, in host populations with highly overdispersed parasite populations, more fish will be infected at much higher levels and thus host mortality may increase. It has been suggested that a decrease in variance to mean ratio with increasing age may be due to parasite induced host mortality (PIHM). By implication, host populations with higher variance to mean ratios are more at risk of suffering PIHM than those with lower variance to mean ratios. The high degree of overdispersion present in the current study and its correlation with chub YCS would suggest that there is a mortality effect in this population. However, confirmation of a demonstrable effect on chub YCS would require calculations of variance to mean ratios in this population post-mortality. Any decrease in the ratios would be suggestive of a mortality effect.

Whilst high variance to mean ratios have been shown to negatively correlated with chub YCS in the River Ouse, regulation of the chub populations by myxozoans, in which lower levels of parasitism are found when population levels are low and are higher when host population numbers increase, appears not to be occurring since myxozoan prevalence and abundance was lowest in 1995 when YCS was high and large numbers of fish were present in the system.

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The validity of the models developed in the present study was, to a degree limited by the small datasets used. However, a clear statistical correlation between YCS, length, relative abundance and disease in 0+ fish was demonstrated. In addition, it should also be noted that the histological method used for data collection provides an underestimate of the number of cysts/parasites present in individual fish. It is clear that Myxobolus spp have a strong effect on chub populations from Rivers Wharfe and Ouse and it is probable that this represents a more general principle (ie parasites of cyprinid fry have an impact on fish length, relative abundance and have a role in determining year class strength). However, for each river system and indeed for each host species, the determining factors probably differ. Synergistic effects between parasite loads, host factors and environmental influences most likely determine the host survivability. Consequently, from the evidence obtained from the studies reported here, population models should incorporate measures of relevant disease status.

Marine fish investigations

A limited number of cases of pathology in marine fish species became available for examination during the period of this contract. A South West fish processing company submitted an example of a rarely reported fungal infection of cod. The infection presented as grey/black discolouration of the musculature.

Histologically, the muscle tissue was necrotic with extensive fungal involvement.

Attempts to culture the fungus from

samples taken from the thawed fillet were unsuccessful and the specific identity of the fungus was not

determined.

Histological assessment of parasites in

Greater pipefish (Syngn athus acus) (n=30) from the Mersey estuary was carried out. A number of different

parasites and prokaryotic infections were recorded including epitheliocystis and Trichodina sp. in

the gills at prevalences of 23% and 13%

respectively, Kudoa cf. quadratum in the skeletal muscle at a prevalence of 3%,

Myxidium cf. incurvatum in the

gall bladder in 33% of the fish, Anisakis simplex in the viscera of 40% of the fish and Cryptocotyle lingua metacercaria in the musculature at a

prevalence of 17%. In addition, an

unidentified digenean was

present in the stomach of

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one fish and cestodes plerocercoids were found in the intestine of two fish. Pathological responses were generally minor and included fibrous encapsulation of digeneans and mild epithelial hyperplasia associated with the presence of trichodinids. No detectable cellular response against the intracellular Kudoa cf. quadratum was seen, a typical feature of infections with many Kudoa spp. In contrast there was significant host response elicited by the Myxidium sp. in the gallbladder. Epithelial hyperplasia and cellular necrosis were consistent findings and in some cases there was atrophy and sloughing of the epithelial cells. The majority of pathologies noted were unlikely to have an impact on host survivability (Longshaw et al., 2004).

A small sample of red band fish (Cepola rubescens) was caught using standard trawl gear on the RV CEFAS Endeavour from an offshore site close to Dundrum Bay, Northern Ireland. Immediately after capture, tails were removed and the remainder of the fish, including head and visceral organs were fixed in either 10% NBF or ethanol. Following return to the CEFAS Weymouth Laboratory, fish were examined for parasites. All organs, including stomach, intestines, liver, gonad, kidney, gall bladder, spleen, gills and eyes were screened for metazoan and protistan parasites.

A total of three parasite species were found including Contracaecum oscalatum (Nematoda) on the viscera and liver (prevalence 100%, abundance 51.3) Anchistrotos onosi (Copepoda) on the skin and gills (prevalence of 100%, abundance 45.38) and unidentified juvenile Digenea in the intestines and stomach (prevalence 100%, abundance 6.23). No protistans, myxozoans, monogeneans, cestodes or acanthocephalans were found within these fish despite specifically searching for them. In addition, 5 fish contained partially digested Caligus sp. in the stomach.

This is, as far as we are aware, the first report of the parasite fauna of red band fish in the Irish Sea. A new host record for the copepod A. onosi is provided. This parasite has previously been reported on five-bearded and four-bearded rocklings. It is clear from the data provided, that these fish have a very depauperate parasite fauna, represented by only three species. This may reflect the lifestyle of red band fish, which tend to live in mud burrows and have a restricted diet composed mainly of pelagic copepods. Transmission of the digeneans and nematodes is presumed to occur through this route. Red band fish are physiologically adapted to survive under the hypoxic conditions of the burrow. Given that A. onosi also occur on the fish in these burrows, it follows that the copepod parasites must also be adapted for these harsh conditions.

Shellfish investigations

Crustacean material was obtained from a variety of sources, including the Defra marine fish disease monitoring cruise from offshore sites and from local more inshore sources. This work was undertaken with the assistance of a placement student during 2003/04. The significant finding of Hematodinium sp. in edible crab (Cancer pagurus) was made following the submission of diseased crab material from Guernsey associated with post capture mortalities during the latter part of 2000. Affected crabs showed marked discolouration and were unmarketable dying before and during vivier transportation. In affected crabs, large numbers of plasmodial and vegetative stages of Hematodinium were present in the haemolymph and there was a depletion of reserve cells in the hepatopancreas. Anecdotal information suggested that large numbers were affected and that at least two fisheries are affected. The infection was reported previously in the early 1980's from a variety of locations around the UK. There remains a paucity of information on the seasonality, transmission and market impact of this disease in edible crab (Stentiford et al., 2002).

A novel intranuclear bacilliform virus (IBV) (Baculovirus-like virus) in the brown shrimp (Crangon crangon) from UK estuaries was identified. The virus causes necrosis of the hepatopancreas and midgut epithelial cells. The nuclei of infected epithelial cells were hypertrophied and contained conspicuous eosinophilic inclusions corresponding to the viroplasm, which caused margination of the nuclear chromatin. Infected cells became necrotic and detached from the adjoining epithelial cells and were expelled into the lumen of the hepatopancreatic tubule or the midgut. Prevalence of up to 100 % at sites in the Clyde estuary and high levels (up to 85 %) at sites in the Forth and Mersey estuaries were identified (Stentiford et al., 2004). In addition, an unidentified yeast pathogen in edible crab Cancer pagurus and velvet swimming crab Necora puber infected with Hematodinium were also observed. Yeast cells were observed intracellularly in circulating haemocytes and free in the plasma. Haemocytic encapsulation of yeast cells was also observed (Stentiford et al., 2003). The yeast appeared to cause a co-infection, presumably due to immunosuppression by the primary pathogen. Similar co-infections with yeast have also been noted in virus-infected Crangon crangon from UK estuaries. A novel haplosporidian infection in approximately 10 % of Carcinus maenas captured from UK English Channel site was identified. Haemolymph appears opaque and creamy and coats internal organs (Stentiford et al., 2004). In addition, a Paramartelia-like parasite in

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Cancer pagurus from UK English Channel site was discovered. The parasite was found in connective tissues and tissue sinuses. Further work is planned on the full identification and description of this parasite.

Samples of scallops from four locations in the Irish Sea were examined for the presence of pathogens as part of an ongoing assessment of scallop stocks around England and Wales. Samples were collected during the regular fish disease monitoring as part of the National Marine Monitoring Programme (NMMP) supported by MWD. Natural stocks of queen scallops (Aequipecten opercularis) from locations off the noth coast of Wales exhibited infections with rickettsia-like organisms (RLO) in the gill (40%) and also harboured microsporean infections in the digestive gland (8%). King scallops (Pecten maximus) from these locations and from cultured stocks in Portland harbour did not reveal the presence of infectious agents. A further sample obtained from Cardigan Bay exhibited similar prevalences of RLO and microsporidiosis. Occasional digenean infections and non-specific haemocyte infiltration were noted, but were not regarded as significant (Feist et al., 2001).

Objective 2. To identify those pathogens detected in exotic fish species that may pose a threat to native stocks.

This aspect of the contract included the examination of imports of cold-water ornamental fish on an opportunistic basis when these were returned to the laboratory by the FHI for spring viraemia of carp virus testing. However, only few samples became available during the first two years of the contract and thereafter changes in operating procedure whereby fish were sampled on site and were not returned to the laboratory. Most samples submitted were goldfish varieties imported into the United Kingdom particularly from Israel, the far East and continental Europe. The majority of parasites found were protistan and included Trichodina sp. and Ichthyobodo necator on the gills and Myxobolus spp. in the gall bladder, gills and musculature. Of the metazoan species found, Gyrodactylus sp. and Dactylogyrus sp. were common on the gills and in one sample of goldfish from the Far East, encysted digenean metacercaria (Centrocestus sp.) were present on the gills. Some pathology was noted associated with these parasites. Occasional renal parasites were found and infections with a Chilodinella- like ciliate were detected in gills. Most imported fish have been treated before transport and were not found to harbour heavy parasite burdens. A number of examples of parasitic infections obtained from this work were published (Longshaw and Feist, 2001). The potential for transmission of the protistan parasites to naive native stocks remains a possibility. However, insufficient material was available to conduct transmission experiments.

A population of the exotic weather loach (Misgurnus anguillicaudatus) that had become established in a large pond in the UK were examined for parasites. Both male and female fish were present and there was evidence based on the size classes and numbers present that hey had successfully bred whilst in this open pond system. All weather loach were removed from the system and returned to CEFAS Weymouth for examination. A total of 30 fish were examined for their parasite fauna, and a further 30 preserved for histological examination. Apart from a single species of Gyrodactylus, no other pathogens were detected, either from the parasitological screen or by histological assessment. The Gyrodactylus sp., found in high abundance, was superficially similar to the exotic G. macracanthus although was easily discriminated form this species on a number of morphological features and when data was compared using principal component analysis. In addition, the parasite was unlike any previously reported on native UK hosts. Whilst there was no evidence that the parasite had spread to native species of fish in the pond, the presence and survival of both the host and its parasite raises concern as it clearly demonstrates that both can adequately survive and establish in UK waters. The ability of the weather loach to out compete native stocks is currently unknown and the impact of the Gyrodactylus sp. on native stocks also remains unknown.

A sample of weather loach being imported from Singapore was examined for parasites. As with the established population reported above, only a single species of gyrodactylid was isolated. This parasite was identified as G. macracanthus which has been reported as a pest of concern in Australia.

Populations of two non-native fish species present in the UK, top mouth gudgeon (Pseudorasbora parva) and sunbleak, (Leucaspius delineatus) were examined for parasites and other pathogens as part of a collaborative Defra funded study with CEH Winfrith on the ecological impact of non-native fish species. The sample of top mouth gudgeon was found to be negative. In contrast, the sunbleak caught in a lake system in southern England however, were heavily parasitised. Parasites isolated included Myxidium rhodei (Myxozoa) in the kidney at a prevalence of 47%, Gyrodactylus sp. (Monogenea) on the skin and fins, Rhabdochona sp. (Nematoda) in all fish examined, Diplozoon paradoxum (Monogenea) on the gills at

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a prevalence of 7% the digenean Diplostomum spathaceum in the eyes of 10% of the fish, Ergasilus briani (Copepoda) on the gills at a prevalence of 13% and Neoergasilus japonicus (Copepoda) on the fins at a prevalence of 43%. The two copepod species are of concern since they are non-native to the UK and are included in the Environment Agencies Category 2 list of parasites. Whilst it is known that both of these parasites are established in the UK, this is the first record on these hosts and it is unclear of the role of sunbleak in transmitting these pathogenic parasites to native hosts (Beyer et al., 2005).

Studies on pathogen interactions between two non-native species, topmouth gudgeon and sunbleak were undertaken in collaboration with Dr Rodolphe Gozlan (CEH Winfrith). Dr Gozlan had previously identified an association between introduction of the topmouth gudgeon to a new water body and the loss of condition and population decline of sunbleak. This was confirmed in a number of laboratory experiments conducted at CEH Winfrith. During the latter part of this contract Dr Gozlan approached CEFAS in order to investigate the possibility that topmouth gudgeon may be acting as a carrier for an unidentified pathogen. Samples for bacteriology, virology, parasitology and histology from 29 sunbleak and 10 topmouth gudgeon from the cohabitation experiments were examined. Histologically, moribund sunbleak showed the presence of systemic pathology characterised by granulomatous lesions (prevalence 61%, n=8). These lesions harboured intracellular organisms which were Gram positive. Results for bacteriology and virology were negative and parasite burdens were similar to those encountered in material examined previously (see above). The nature of the granulomatous lesions was similar to those seen in farmed North American salmonids, including Atlantic salmon affected by ‘Rosette agent’ (Sphaerothecum destruens) (Arkush et al., 2003). Molecular studies using primers for a segment of the 18s ribosomal DNA of S. destruens confirmed the presence by PCR of this agent in moribund sunbleak at 67% prevalence (n=12) but also in overtly unaffected fish from the exposed group at lower prevalence (28%, n=32). DNA sequences from positive bands were compared to sequences submitted to GenBank and EMBL databases and were found to exhibit 100% homology to salmonid S. destruens. All P. parva tested by PCR were negative. However, the difficulty in detecting carrier status using conventional diagnostic tests is well recognised. These results indicate that the non-native topmouth gudgeon acts as healthy carrier for S. destruens and that the pathogen is able to infect other cyprinids (Gozlan et al., 2005). Further investigations are required in order to understand the full implications of this finding for native fish species.

Objective 3. To improve knowledge of the normal structure, histology and parasite fauna of marine fish species of potential for aquaculture.

This objective became low priority after year two and was dropped from the programme in order to concentrate resources to Objective 1. However, material was obtained from three species, namely cod (Gadus morhua), haddock (Melanogrammus aeglifinus) and turbot (Scophthalmus maximus) from the environmental monitoring cruise (contract AE003). All major organs and tissues were sampled and processed for histological examination. This material was archived in the RAP for future reference as required.

Objective 4. To establish the Registry of Aquatic Pathology (RAP) as an internationally recognised resource and depository for pathological specimens and parasites.

The Registry of Aquatic Pathology (RAP) database was developed to facilitate access to a large number of pathological specimens accumulated over many years at CEFAS Weymouth Laboratory. The collection consists of histological slides and stained tissue smears as well as wax embedded blocks and some fixed specimens. During the period of this contract the collection was significantly expanded to include parasite specimens. Although the emphasis has been given to accessions for pathological conditions, the collection also contains ‘normal’ tissues from a variety of fish, molluscs and crustacea. The conversion to a database format has allowed the inclusion of digital images of specimens to be incorporated with the accessions. Currently, the RAP contains over 800 accessions.

For each accession, information on the host species (common and Latin name), origin (geographic and specific location, e. g. fish farm), donor, aetiology, significance and a description of the pathology or tissue is provided. The database can be interrogated using several search parameters, including by condition, individual accession number, submitter or using key words (see below).

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In addition, search by RAP categories (salmonids, cyprinids, shellfish, parasites etc.) or species name is possible.

Each individual accession entry contains the specific data related to that specimen as well as additional information such as published references on the subject or papers that refer to the accession itself.

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For most histological specimens, selected images have been taken of the pertinent features of the pathology and/or pathogen. Where appropriate, several images at different magnification are included.

The RAP is continually updated as new scientific information becomes available and as new specimens are incorporated. The value of the RAP as a resource for training and reference is also continually increasing and has already been used to fulfil requests to CEFAS (as OIE Reference Laboratory for SVC) to provide material showing SVC pathology. Accessions were also used to provide reference material for the EU Community Reference Laboratory (IFREMER, La Tremblade, France) on scallop pathology. It is recognised that access to the RAP database and collection to the wider scientific community is essential in order to obtain maximum benefit for training, research and diagnostic purposes as well as for the continued submission of material from scientists involved in fish and shellfish pathology and parasitology.

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To this end it is anticipated that the RAP will be made available via the OIE collaborating centre website. Work on the RAP will be continued under FC1166

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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Feist, S. W., Lohrmann, K. B. and Brand, A. R. (2001) Disease survey on the scallops Pecten maximus (L.) and Argopecten opercularis (L.) in UK waters. Proceedings of the XIII International Pectinid Workshop. Coquimbo, Chile. 18 -24 April 2001. Longshaw, M., Feist, S. W., Matthews, R. A & Figueras, A. (2001) Ultrastructural characterisation of Marteilia species (Paramyxea) from Ostrea edulis, Mytilus edulis and Mytilus galloprovincialis in Europe. Diseases of Aquatic Organisms, 44, 137-142.Longshaw, M. & Feist, S.W (2001) "Parasitic Diseases" p. 167-183 In: BSAVA Manual of Ornamental Fish (Ed. W.H. Wildgoose) BSAVA.Ramsay, K., Bergmann, M., Veale, L. O., Richardson, C. A., Kaiser, M. J., Vize, S. J. & Feist, S. W. (2001) Damage, autotomy and arm regeneration in starfish caught by towed demersal fishing gears. Marine Biology, 138, 527-536.Stentiford, G. D. (2001) Hematodinium in Norway lobsters. Shellfish News, 11, 16-17. Lohrmann, KB, Brand, AR & Feist, SW (2002) Comparison of the parasites and pathogens present in a cultivated and in a wild population of scallops (Argopecten Purpuratus Lamarck, 1819) in Tongoy Bay, Chile. Journal of Shellfish Research 21: 557-561.Stentiford, GD, Green, M, Bateman, K, Small, HJ, Neil, DM & Feist, SW (2002). Infection by a Hematodinium-like parasitic dinoflagellate causes Pink Crab Disease (PCD) in the edible crab Cancer pagurus. Journal of Invertebrate Pathology 79, 179-19.Stentiford, GD (2002). Pink Crab Disease in edible crabs. Shellfish News 13, 16-17.Longshaw, M., Pursglove, M. & Shinn, A. P. (2003) Gyrodactylus quadratidigitus n. sp. (Monogenea: Gyrodactylidae), a parasite of the leopard-spotted goby Thorogobius ephippiatus (Lowe) from the south-western coast of the UK. Systematic Parasitology 55(2): 151-157.Stentiford, G.D., Evans M., Bateman, K., Feist, S.W. (2003). Co-infection by a yeast-like organism in Hematodinium-infected European edible crabs (Cancer pagurus) and velvet swimming crabs (Necora puber) from the English Channel. Diseases of Aquatic Organisms 54, 195-202.Stentiford, G.D, Longshaw, M., Lyons, B.P., Jones, G., Green, M. & Feist, S.W. (2003) Histopathological biomarkers in estuarine fish species for the assessment of biological effects of contaminants. Marine Environmental Research 55: 137-159 Feist, S.W., Longshaw, M., Hurrell, R.H. & Mander, B. (2004) Observations of Dermocystidium sp. infections in bullheads (Cottus gobio L.) from a river in southern England. Journal of Fish Diseases, 27,225-231.Longshaw, M., Green, M.J. & Feist, S.W. (2004) Histopathology of parasitic infections in greater pipefish (Syngnathus acus) from an estuary in the UK. Journal of Fish Diseases, 27, 245-248.Stentiford, G.D., Feist S.W., Bateman, K.S., Hine P.M. (2004) A haemolymph parasite of the shore crab Carcinus maenas: pathology, ultrastructure and observations on crustacean haplosporidians. Diseases of Aquatic Organisms, 59, 57-68.Stentiford, G.D., Bateman, K., Feist, S.W. (2004) Pathology and ultrastructure of an intranuclear bacilliform virus (IBV) infecting brown shrimp Crangon crangon (Decapoda: Crangonidae). Diseases of Aquatic Organisms, 58, 89-97.Stentiford, G.D. & Feist, S.W. (2005) A histopathological survey of shore crab (Carcinus maenas) and brown shrimp (Crangon Crangon) from six estuaries in the United Kingdom.Journal of Invertebrate pathology, 88, 136-146.Stentiford, G.D., Bateman, K., Feist, S.W. (2004). A virus infection in European brown shrimps (Crangon crangon). Shellfish News, May 2004.Canning, E. U., Feist, S. W., Longshaw, M., Okamura, B., Anderson, C. L., Tsuey Tse, M. & Curry, A. (2005) Microgemma vivaresi n. sp. (Microsporidia, Tetramicridae), infecting liver and skeletal muscle of sea scorpions, Taurulus bubalis (Euphrasen, 1786) (Osteichthys, Cottidae), an inshore littoral fish. Journal of Eukaryotic Microbiology, 52(2), 123-131.Feist, S. W. & Longshaw, M. Phylum Myxozoa. In 'Fish Diseases and Disorders' Volume 1: Protozoan and Metazoan Infections (ed. P. T. K. Woo). (Submitted).Stentiford, G.D., Shields, J.D. (2005) A review of the parasitic dinoflagellates Hematodinium species and Hematodinium-like infections in marine crustaceans. Diseases of Aquatic Organisms (in press).Gozlan, R. E., St-Hilaire, S., Feist, S. W., Martin, P. & Kent, M. L. (2005) An emergent infectious disease threatens European fish biodiversity. Nature (in press).Beyer, K., Kochanowska, D., Longshaw, M., Feist, S.W. & Gozlan, R.E. (2005) Invasive sunbleak, a new host for a non-native parasite in Europe. Journal of Fish Biology (in press)

Presentations

A total of 17 presentations were made at interational conferences including the XIII International Pectinid Workshop, the British Society for Parasitology Spring meeting, the European Association of Fish Pathologists conference, the International crustacea Congress V, the 'Myxozoan Symposium in Nanaimo and the International Congress of Parasitology.

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