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Evidence Project Final Report

 Note In line with the Freedom of Information Project identification Act 2000, Defra aims to place the results of its completed research projects in the public domain wherever possible. 1. Defra Project code FC1186 (C3390) The Evidence Project Final Report is designed to capture the information on 2. Project title the results and outputs of Defra-funded Susceptibility of European Crustaceans to EC Directive- research in a format that is easily listed pathogens publishable through the Defra website An Evidence Project Final Report must be completed for all projects. 3. Contractor  This form is in Word format and the organisation(s) boxes may be expanded, as appropriate.  ACCESS TO INFORMATION The 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 54. Total Defra project costs £ 342,330 project. Defra may also disclose the (agreed fixed price) information to any outside organisation

acting as an agent authorised by Defra to 5. Project: start date ...... 1/4/2009 process final research reports on its behalf. Defra intends to publish this form on its website, unless there are strong end date ...... 31/3/2012 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.

EVID4 Evidence Project Final Report (Rev. 06/11) Page 1 of 23 6. It is Defra‟s intention to publish this form. Please confirm your agreement to do so...... YES x NO (a) When preparing Evidence Project Final Reports 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 Evidence Project Final Report 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 Summary 7. 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.  EC Council Directive 2006/88 lists three crustacean diseases: White Spot Disease (WSD) caused by the White Spot Syndrome Virus (WSSV), Yellowhead disease (YHD) caused by Yellowhead Virus (YHV) and Taura syndrome (TS) caused by Taura syndrome virus (TSV). WSD is currently listed as „non-exotic‟ within the EU based upon its reported occurrence in penaeid shrimp farms in the Mediterranean (Stentiford and Lightner, 2011).  Very little previous work has been conducted on the susceptibility of non-tropical crustacean hosts to the listed pathogens. Furthermore, few guidelines exist for the confirmation of „susceptibility‟ per se, or the definition of „relative susceptibility‟ of given host species. In the context of animal and product trading, this has led to knowledge gaps in risk assessments concerned with potential transboundary movement of infectious disease agents in live animals and commodity.  The current study has provided a framework for the assessment of susceptibility in decapod crustacean hosts to the listed pathogens (WSSV, TSV and YHV) and has tested this framework on a range of commercially- and ecologically-important crustacean hosts found within UK lakes, rivers and seas (Stentiford et al. 2009). Further, it has defined the profile of disease in those hosts deemed to be susceptible and has made this information available via publication in peer- reviewed articles (Bateman et al. 2012a).  Disease challenge protocols for non-model crustacean hosts (crabs, lobsters, crayfish, shrimp) collected from natural habitats were developed and optimised through the current project. Protocols included viral challenge to cold water lobsters (Nephrops norvegicus), brackish water crabs (e.g. Chinese mitten crabs, Eriocheir sinensis) and freshwater crayfish (e.g. white clawed crayfish Austropotamobius pallipes). These protocols are available for use in future studies of this type.  All crustacean hosts tested herein were considered susceptible to infection with WSSV, though the outcome of infection ranged considerably from asymptomatic carrier (e.g. the shore crab Carcinus maenas), to highly susceptible (e.g. juvenile life stages of the European lobster, Homarus gammarus). We were able to define the pathological profile of WSSV infection and disease in these hosts and to compare this to the OIE defined profile for infection and disease associated with WSSV in farmed penaeid shrimp. Hosts were classified for the first time into relative levels of susceptibility (Bateman et al. 2012a).  Three crustacean host species (signal crayfish Pacifastacus leniusculus, shore crab C. maenas, and lobster H. gammarus) were considered as potentially susceptible to infection with TSV though in all cases, infection did not progress to disease, defining all of these hosts as asymptomatic

EVID4 Evidence Project Final Report (Rev. 06/11) Page 2 of 23 carriers for TSV. Further work will be required to define the pathological profile of TSV infection within these hosts and to confirm long-term carrier capacity.  Environmental manipulation (temperature) designed to test potential for shift from asymptomatic carrier status to disease, was tested in a low-susceptible host (C. maenas). Here, ambient elevated temperatures for extended time periods following exposure to WSSV via injection did not have a significant effect on disease induction, suggesting that at least for some UK species, temperature is not the limiting factor in pathogenesis of WSSV. In other hosts (H. gammarus), exposure to WSSV at low (winter) temperatures led to infection, with higher temperatures required for induction of disease. These trials support the notion that the outcome of exposure (to WSSV) differs considerably between host species and further that susceptible hosts are found across marine, brackish and freshwater habitats within the UK.  To assist with knowledge gaps in Import Risk Assessments (IRA) for tropical shrimp commodity products, an exposure trial using lobsters (H. gammarus) to supermarket shrimp originating in Asia and Latin America demonstrated the presence and viability of WSSV in these products. Feeding bioassays to lobsters led to infection following a single feed on WSSV positive shrimp tails. This is the first demonstration of infection of a temperate water crustacean following consumption of low viral load supermarket shrimp commodity (Bateman et al. 2012b). Data provided here provides direct evidence for widespread WSSV-contamination of tropical shrimp in UK supermarkets and the potential for this product to infect local crustacean fauna.  Several areas for future research have been identified from the current work programme. These include 1. To decipher the genetic basis for relative resistance to WSSV between different hosts and to utilise this data when assessing risk of trade. 2. To understand the role of commodity products in the translocation of crustacean pathogens into the UK/Europe, and inform IRAs on the consequence of their diversion into aquatic habitats (e.g. in angling baits). 3. To investigate the susceptibility of UK hosts to other OIE listed pathogens likely present in commodity arising from tropical locations. 4. To understand the interaction of exotic pathogens with native pathogens already infecting local host species. 5. To investigate the potential for passage of WSSV (and other viruses) from UK host to UK host within the context of consequence assessment for the establishment of these pathogens in UK waters.

Project Report to Defra 8. 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 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 Exchange).

EVID4 Evidence Project Final Report (Rev. 06/11) Page 3 of 23

Overview

EC Council Directive 2006/88 (herafter „the Directive‟) lists three crustacean diseases: White Spot Disease (WSD) caused by the White Spot Syndrome Virus (WSSV), Yellowhead disease (YHD) caused by Yellowhead Virus (YHV) and Taura syndrome (TS) caused by Taura syndrome virus (TSV). WSD is currently listed as „non-exotic‟ within the EU based upon its reported occurrence in penaeid shrimp farms in the Mediterranean (Stentiford and Lightner, 2011). YHD and TS are listed as „exotic‟ due to their apparent absence from the EU. The inclusion of these viral diseases within the Directive recognises their global importance in causing massive economic losses in farming regions, the lack of control measures available to deal with outbreaks and the potential for them to occur in non-farmed hosts, either as passive, latent or disease causing agents.

To date, despite the relatively free transfer of these viral agents around the globe, either with the movement of live animals for farming (e.g. broodstock and larvae) or as contaminating agents in commodity products (e.g. frozen shrimps) and their demonstrable ability to establish in new hosts at point of destination, very little research has been carried out on the susceptibility of European species to these disease causing agents. Furthermore, little information is available on the environmental tolerance of these viral agents or their ability to establish infection and disease in non-target hosts should they be introduced. Available data is especially lacking for European crustaceans, particularly those that exist at temperatures that may be considered out-with the normal range experienced by these viruses in endemic zones in Asia and South America.

Crustaceans are keystone elements of all aquatic systems. They are fundamental elements in the food chain and as such form a significant element of the diet of most fish species. In fisheries terms, their importance in European marine waters is amply demonstrated by a total crustacean fishery production of c.400 thousand tons per annum, with a large majority of this comprising the decapods, shrimp (c. 200kt), lobsters (c. 60kt) and crabs (c. 85kt) - a significant proportion of this concentrated in the shallow shelf seas around the UK. In UK freshwaters, the white claw crayfish (Austropotomobius pallipes), is considered endangered and is protected under UK and European legislation (including the IUCN Red Data list, the Wildlife and Countryside Act 1981 and the EC Habitats Directive 1992). A significant feature of the viral pathogen WSSV is its potentially wide host suscpetibility range (listed as 'all decapods' by the OIE). When coupled to apparent environmental resiliance, the wide host range identifies it as a significant potential threat to sustainability of UK crustacean stocks in freshwater and marine environments.

The current programme aimed to gather fundamental data to assess the susceptibility of key UK crustacean wildlife and fishery species to pathogens listed in the Directive. In addition, to address the potential for environmental variables to affect susceptibility and pathogenesis in a European context and to inform aligned risk assessments on the potential for WSD, YHD and TS to establish in the UK aquatic environment following an introduction event. The programme has also supported a thorough review of existing literature on susceptibility to the listed pathogens (published in Stentiford et al. 2009) – this review informing the subsequent direction of experimental trials carried out herein.

Scientific Objectives

The scientific objectives for FC1186 were as follows:

1. To define taxonomic susceptibility range to WSD, TS and YHD among ecologically and economically important UK freshwater, brackish and marine crustacean species. 2. To define possible transmission modes for WSD, TS and YHD (via feeding, injection, cohabitation, water) to UK hosts, identify initial infection sites within susceptible hosts and document. pathogenesis of disease in hosts residing within the UK ambient water temperature range 3. To define ambient temperature range for promotion of „carrier‟ and „disease‟ status of WSD, TS and YHD in selected model UK crustacean hosts. 4. To demonstrate pathogen viability when established in UK hosts by onward transmission to known susceptible tropical crustacean hosts (Specific Pathogen Free Penaeus vannamei). 5. To collate data on susceptibility, transmission and environmental variables to inform risk assessment for establishment of WSD, TS and YHD in UK crustacean hosts.

The delivery against specific objectives will be presented and discussed in the following text and figures. Detailed analyses of the main findings published to date can also be found in Bateman et al. (2012a), Bateman et al. (2012b) and Stentiford et al. (2009).

EVID4 Evidence Project Final Report (Rev. 06/11) Page 4 of 23 Approach and findings

1. Defining the susceptibility range, transmission pathways and viability of UK crustacean hosts to the listed pathogens (Objectives 1, 2 and 4).

1.1 Literature review

A thorough literature review initiated at the outset of the project coincided with liaison between the PI and working groups of the European Food Safety Authority (EFSA). The outcome was a definition of criteria to support evidence for „susceptibility‟ of a given host species to those pathogens listed in the Directive. These criteria (as published in Stentiford et al. 2009) are provided in Table 1.

Table 1

In terms of supporting international trade whilst protecting farmed and wild crustacean species within European aquatic habitats, it is important to define which host species, particularly those that are traded, are susceptible to the listed diseases since these may pose a risk of transporting the pathogen into the UK via normal trade routes. According to the Directive, a susceptible species is one that is primarily susceptible to infection with or without disease manifestation. As such, it can support replication of an agent or an infestation, which may lead to the development of disease. In terms of the review by Stentiford et al. (2009) and the working groups of EFSA, it was also important to consider susceptibility in the context of i) „natural pathways‟ provided by the experimental design utilised in reported studies, ii) compliance with the four objective criteria pertaining to host susceptibility (see Table 1, above), and iii) thorough identification of the causative agent (usually by applying OIE confirmatory tests). These criteria were applied in all experimental challenges carried out within FC1186. Importantly, the susceptibility criteria listed in Table 1 enable discrimination of actual infection from mechanical vector status of a host. For example, in relation to the viral pathogens causing WSD, TS and YHD, evidence of replication (criterion A) can be provided by the presence of characteristic inclusion bodies, RNA [for the DNA virus causing WSD], and TEM demonstration of virus particles. In the absence of crustacean cell lines, the presence of a viable organism (criterion B) may be inferred from successful transmission from the species of interest to specific pathogen free (SPF) susceptible hosts. Detection of characteristic clinico-pathologic changes (criterion C) and the anatomic location of the pathogen (criterion D) are important features when excluding potential passive contamination of the host. These criteria can be addressed by techniques such as histology, immuno-histochemistry (IHC), or in-situ hybridisation (ISH) or alternatively, target organ dissection and specific detection using techniques such as quantitative PCR (QPCR). In the case of WSD, TS and YHD, a species would be regarded as infected and therefore susceptible by interpretation of combinations of A, B C, and D. Stentiford et al. (2009) suggested that criterion B alone (presence of viable organism) would not be enough to identify a species as susceptible because it does not exclude mechanical contamination. In these cases, „hosts‟ may be assigned vector status.

1.11 What did the review tell us about susceptibility to WSD, TS and YHD?

As written, the Directive lists „all decapods‟ (c. 22,000 extant species of shrimp, prawn, crayfish, crab and lobster) as susceptible to WSD. In the review by Stentiford et al. (2009), published evidence was only available for a total of 98 potential susceptible species or genera with scientific data (Table 1) only available for 67 of these. Numerous other aquatic organisms, including rotifers, bivalves, polychaete worms, and non- decapod crustaceans, copepods, amphipods, isopods, and several aquatic insects were classified as potential mechanical vectors. In fact, any insect or living organism present in a WSSV infected pond may become a mechanical vector of the disease by surface or gut contamination with the viral particles. For TS the Directive lists Gulf white shrimp (Penaeus setiferus), Pacific blue shrimp (P. stylirostris), and Pacific white shrimp (P. vannamei) as species susceptible to infection with the TS virus. In addition, Stentiford et al. (2009) provided evidence for ten additional susceptible host species from the scientific literature. These included predominantly penaeid shrimp species (Penaeus vannamei, P. duorarum, P. monodon, P.

EVID4 Evidence Project Final Report (Rev. 06/11) Page 5 of 23 setiferus, P. chinensis, P. stylirostris, P. aztecus, Metapenaeus ensis, with some evidence for Penaeus schmitti, and P. japonicus). For YHD, the Directive lists Gulf brown shrimp (Penaeus aztecus), Gulf pink shrimp (P. duorarum), Kuruma prawn (P. japonicus), black tiger shrimp (P. monodon), Gulf white shrimp (P. setiferus), Pacific blue shrimp (P. stylirostris), and Pacific white shrimp (P. vannamei) are susceptible to infection with YH virus. In addition, Stentiford et al. (2009) provided evidence for eighteen additional susceptible host species from the scientific literature. Once again, these predominantly included penaeid shrimp species (Penaeus monodon, P. merguiensis, P. vannamei, P. setiferus, P. aztecus, P. duorarum, Metapenaeus brevicornis, M. affinis, P. esculentus, P. schmitti, P. japonicus, M. ensis, and M. bennettae) plus the palaemonid shrimps Macrobrachium lanchesteri and Palaemon styliferus.

There is a widely divergent taxonomic range of host susceptibility to the three crustacean diseases listed in the Directive (Stentiford et al. 2009). In contrast to the apparently rather limited susceptible host range for TS and YHD (predominantly hosts in the family Penaeidae), susceptibly to WSD was demonstrated for hosts in both large suborders (Dendrobranchiata and Pleocyemata) of the Decapoda. This remarkable higher-level taxonomic diversity in WSD susceptibility was demonstrated for 3 families (Penaeidae, Solenoceridae, Sergestidae) of the 7 families in the Suborder Dendrobranchiata; and further 24 families of the 94 families comprising the various Infraorders and Superfamilies of the Suborder Pleocyemata. The latter include susceptible species in the 5 of the 8 decapod infraorders: Anomura (e.g. hermit crabs), Astacidea (e.g. clawed lobsters, freshwater crayfish), Brachyura (crabs), Caridea (shrimps), Palinura (e.g. slipper lobsters). To date, susceptibility has not been demonstrated experimentally for the infraorders Palinuridea (spiny lobsters), Stenopodidea (e.g. cleaner shrimps) and Thalassinidea (e.g. ghost shrimps). Overall, WSD shows one of the highest susceptible species ranges of any known virus.

This review is of direct use in informing risk assessment for the importation of potentially susceptible hosts and in contrast to use of fixed and rapidly outdating lists of susceptible species to a given pathogen (such as WSSV), it also provides a framework for considering „taxonomic spread‟ in host range as an alternative approach to assessing susceptibility – this more in line with the variation in virulence strategies for the pathogens (including those listed for fish and molluscs) in the Directive. In addition, the concept allows for a precautionary principle to be applied to those species within a potentially susceptible taxonomic group (e.g. genus, family, infraorder) that for various reasons have never been rigorously tested for susceptibility. Finally, it has provided a robust framework for the subsequent experimental work carried out within the remainder of project FC1186, on the susceptibility of EU crustaceans to pathogens listed in the Directive.

1.12 Susceptibility of EU crustaceans to the listed pathogens: UK perspective

The review of Stentiford et al. (2009) included several susceptible species present in European marine, brackish and freshwater habitats. These species include those considered as economically important (farmed or fished), ecologically important (e.g. protected under international legislation), or incidental (no current economic value and not protected by legislation). The review considered these resident species types to propose that EU Member States can be broadly categorised into 3 types; Type 1 states possess coldwater marine borders, estuaries and freshwaters (e.g. Northern Europe, including the UK); Type 2 states possess warmer water marine borders, estuaries and rivers (e.g. Mediterranean); and Type 3 states are landlocked and only contain freshwaters (e.g. Central and Eastern Europe). Each type may or may not support commercially significant susceptible species (e.g. lobsters, crabs, crayfish) and ecologically significant susceptible species (e.g. crayfish), while all types are likely to contain susceptible incidental species (at least to WSSV).

For Type 1 states (e.g. UK), the review by Stentiford et al. (2009) mentioned several species known to be susceptible to WSSV from marine (shore crab (Carcinus maenas), edible crab (Cancer pagurus), swimming crab (Liocarcinus depurator), velvet crab (Liocarcinus puber) and brown shrimp (Crangon crangon) and freshwater habitats (the crayfish: Pacifastacus leniusculus, Astacus leptodactylus and Orconectes limosus). Furthermore, given the fact that susceptibility to TS and YHD is somewhat restricted to the penaeids, Type 1 (such as the UK) are likely at greatest risk from the introduction of broader range pathogens such as WSSV. This contrasts the scenrario in Type 2 states (e.g. Spain), where additional susceptible marine species include farmed and wild penaeid shrimp species (e.g. P. japonicus, P. kerathurus, P. semisulcatus, Metapenaeus spp.). In these states, farmed and wild stocks may be potentially at risk from introduction of the penaeid-oriented pathogens YHD and TS, in addition to the broader range pathogen causing WSD. Finally Type 3 states (containing only freshwaters) will only support crayfish species and other susceptible incidental hosts and as such, introduction of WSD poses the greatest risk in these cases.

This Member State level risk-ranking (based upon susceptible species and habitats) was utilised to carry out a range of experimental exposures of key UK crustacean hosts from marine, brackish and freshwater habitats to the listed pathogens. Due to the fact that the review demonstrated highest risk from the pathogen WSSV, trials were focussed primarily on this pathogen. However, despite the scientific evidence for penaeid-level susceptibility to TS and YHD, several separate exposure trials of UK crustaceans to the

EVID4 Evidence Project Final Report (Rev. 06/11) Page 6 of 23 listed pathogens TSV and YHV were also carried out. A summary of all challenges carried out under FC1186 is provided in Table 2.

1.2 Susceptibility testing in UK crustacean hosts

1.2.1 White Spot Syndrome Virus (WSSV): exposure trial design

The results from susceptibility testing for WSSV in UK crustacean hosts is presented in full in the peer- reviewed publication of Bateman et al. (2012a). Susceptibility to WSSV infection was tested in seven non- model decapod species commonly found in European marine, estuarine and freshwater habitats. Susceptibility was assessed using criteria developed by the European Food Safety Authority (EFSA) and summarised by Stentiford et al. (2009). For WSD, the specific characteristics utilised to test the four criteria (replication, viability, pathology and location) are given in Table 1. These characteristics were assessed in each of the non-model decapod species following their exposure to WSSV. Challenge protocols (as outlined below) were developed as part of project FC1186 since standardised trials to non-model species are not widely publicised in the literature.

Samples were collected from natural sources as follows: a total of 18 edible crabs (Cancer pagurus) and 18 European lobsters (Homarus gammarus) were captured using baited pots in the Weymouth and Portland area of the English Channel, United Kingdom (5032‟50‟‟N 002º11‟00‟‟W). Sixty Norway lobsters (Nephrops norvegicus) were captured on the RV Cefas Endeavour from the North Sea fishery (5408‟566‟‟N 002º35‟019‟‟E) using a Granton trawl and transported to the laboratory in tanks with a running sea water supply. Fifteen White claw crayfish (Austropotamobius pallipes), were sourced from a breeding facility in Stainforth (Near Settle) Yorkshire, United Kingdom prior to transportation to the laboratory in a small amount of freshwater with an air supply. Sixty Signal crayfish (Pacifastacus leniusculus) were sourced from a crayfish farm in Kent, United Kingdom and transported back to the laboratory overnight. Sixty Chinese Mitten Crab (Eriocheir sinensis) were captured using baited pots from the River Thames, London, United Kingdom (51˚27‟12‟‟N, 00˚00‟44‟‟E) and transported back to the laboratory. Sixty shore crabs (Carcinus maenas) were collected from the shoreline at Newton‟s Cove, Weymouth, UK (50o34‟ N, 02o22‟ W) or using drop-nets in Weymouth Harbour (50°36.6' N, 02°27' W). Trials occurred over the duration of the project and were not carried out simultaneously.

All experimental trials were performed within the biosecure exotic disease facility at the Cefas Weymouth laboratory and utilised local, filtered and UV treated water. Day length was set at 14 h/day, night was at 10h with a 30 min fade to simulate dusk and dawn. Flow rate was set a 3-4 l/min and for marine species, salinity during the experimental period remained constant at 35ppt. Temperature was regulated according to the experimental conditions required. All animals utilised in experimental challenge trials appeared externally healthy. Species-specific challenges using WSSV were carried out independently (no multi-species challenges). Animals were transferred into custom-made compartments within large trough tanks, with individuals separated by tank divisions to prevent conflict but sharing the same water supply. Water temperatures in all tanks was held constant at 20°C for C. maenas, E. sinensis, A. pallipes and P. leniusculus, and 16°C for N. norvegicus. The different challenge temperature utilised for these species was chosen to reflect the maximal summer temperatures likely experienced by these species in Europe. For C. pagurus and H. Gammarus, chelipeds were banded to prevent conflict prior to their transfer to two large tanks (n=6 per tank) and three medium tanks which had been divided in half (n=2 per tank). Water temperatures in all tanks was held constant at 20°C for C. pagurus and at 15°C for H. gammarus. Once again, ambient temperatures were chosen to reflect maximal summer temperatures likely experienced by these species in Europe. All animals were acclimatised to these exposure trial condition temperatures for a minimum of one week before trials commenced. Examples of holding conditions for the various test species are given in Fig. 1.

EVID4 Evidence Project Final Report (Rev. 06/11) Page 7 of 23

Fig. 1. Experimental trial holding conditions for a representative range of crustacean species tested for susceptibility to WSSV. (A) Adult Nephrops norvegicus in flow-through trough compartments (B) Juvenile Austropotamobius pallipes in „Orkney pots‟ (C) Adult Carcinus maenas in flow-through trough compartments. (D) Juvenile Homarus gammarus in „Orkney pots‟ (E) Adult Austropotamobius pallipes in pipe shelters. (F) Adult Penaeus vannamei in plastic pots.

Viral inoculates of WSSV were obtained from the OIE reference laboratory for White Spot Syndrome Virus (WSSV) at the University of Arizona, USA. The OIE isolate of WSSV (UAZ 00-173B) was generated in Penaeus vannamei (Pérez Farfante & Kensley, 1997) from an original outbreak of WSD in Penaeus chinensis in China in 1995. Subsequent passage of this isolate into Specific Pathogen Free (SPF) P. vannamei held at the Cefas Weymouth laboratory have demonstrated continued virulence of this isolate (data not reported here). All challenges reported utilised WSSV-infected P. vannamei carcasses generated within the Cefas Weymouth laboratory. As such, WSSV-infected shrimp carcasses were prepared by injection of the UAZ 00-173B isolate into SPF P. vannamei obtained from the Centre for Sustainable Aquaculture Research (CSAR) at the University of Swansea, United Kingdom. Individual P. vannamei were inoculated via intra-muscular injection of the diluted viral homogenate at a rate of 10 l g-1 shrimp weight. Following incubation, dead and moribund shrimp were removed from the experimental tanks and infection with WSSV was confirmed using histopathology, transmission electron microscopy (TEM) and PCR as appropriate (see below for techniques). Remaining tissues were stored at –80oC until required. Confirmed infected and uninfected (sham-injected) carcasses were used to prepare inoculums and feed rations for challenge studies using the non-model host species. Infected and uninfected shrimp carcasses were macerated in isolated conditions using a sterile razor blade prior to homogenisation in sterile saline (4ml of saline per gram of minced tissue) using a blender until tissues were liquefied. The homogenate was centrifuged at 5,000 x g for 20 minutes at 4°C to pellet solid debris prior to the supernatant being diluted 1:20 with sterile saline and filtered to form the inoculums for the injection studies. For feeding trials, confirmed infected and uninfected carcasses were macerated into approximately 2-3 mm3 blocks using a sterile razor blade immediately prior to feeding.

For all exposure trials, a similar protocol was followed. Group 1 (negative control feed) animals were fed with a single ration of confirmed SPF shrimp tissue at approximately 5% bodyweight on Day 0 of each trial. Group 2 (WSSV positive feed) animals were fed with a single ration of confirmed WSSV-infected (but otherwise SPF) shrimp tissue at a ratio of approximately 5% bodyweight on Day 0 of each trial. Group 3 (WSSV positive injection) animals were injected with a single dose of the diluted WSSV homogenate (see above) at a rate of 10µl g-1 wet body weight, on Day 0 of each trial. Since the key objective of this study was to investigate the potential for WSSV infection via a confirmed natural route (i.e. feeding), and the fact that we do not consider direct injection as a natural route of infection for WSSV (see Stentiford et al., 2009), Group 3 served purely to introduce WSSV into hosts for the investigation of histopathological progression of disease in non-model decapods. As such, a negative control (sham-injected) group was not included in the trials. Thereafter, samples in all tanks were fed on squid tissues at a ratio of approximately 3-4% wet body weight.day-1 for the remainder of the trial period. Tanks were observed regularly throughout daylight hours. Dead and terminally morbid samples were removed from each tank and dissected. At the end of each challenge trial (Day 10), and for moribund animals sampled within the trial, surviving animals were chilled on ice for 30 minutes prior to dissection. As standard, gill, epidermis, hepatopancreas, heart, gonad, nerve and muscle were placed into histological cassettes and fixed immediately in the appropriate fixative. Davidson's fixative made with seawater (Davidson‟s seawater) was used for tissues from marine species

EVID4 Evidence Project Final Report (Rev. 06/11) Page 8 of 23 and Davidson‟s fixative made with tap water (Freshwater Davidson‟s) was used for all freshwater crayfish tissues. For molecular analyses, gill, epidermis and hepatopancreas samples were removed and placed into tubes containing 100% ethanol. For electron microscopy, gill, epidermis, hepatopancreas and heart tissues were fixed in 2.5% Glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) for EM. The remaining tissues and carcasses were frozen at -80°C for storage.

For histology, fixation was allowed to proceed for 24 h before samples were transferred to 70 % industrial methylated spirit. Fixed samples were processed to wax in a vacuum infiltration processor using standard protocols. Sections were cut at a thickness of 3-5 µm on a rotary microtome and mounted onto glass slides before staining with haematoxylin and eosin (H&E) and Feulgen stains. Stained sections were analysed by light microscopy (Nikon Eclipse E800) and digital images and measurements were taken using the Lucia™ Screen Measurement System (Nikon, UK). For electron microscopy, tissues were fixed in 2.5 % glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) for 2 h at room temperature and rinsed in 0.1 M sodium cacodylate buffer (pH 7.4). Tissues were post-fixed for 1 h in 1 % osmium tetroxide in 0.1 M sodium cacodylate buffer. Samples were washed in three changes of 0.1 M sodium cacodylate buffer before dehydration through a graded acetone series. Samples were embedded in Agar 100 epoxy (Agar Scientific, Agar 100 pre-mix kit medium) and polymerised overnight at 60ºC in an oven. Semi-thin (1-2 µm) sections were stained with Toluidine Blue for viewing with a light microscope to identify suitable target areas. Ultrathin sections (70-90 nm) of these areas were mounted on uncoated copper grids and stained with 2 % aqueous uranyl acetate and Reynolds‟ lead citrate (Reynolds, 1963). Grids were examined using a JEOL JEM 1210 transmission electron microscope and digital images captured using a Gatan Erlangshen ES500W camera and Gatan Digital Micrograph™ software.

For PCR, Genomic DNA was extracted from tissues using a High Pure PCR Template Preparation Kit (Roche) following the manufacturer‟s protocols. Ethanol-preserved tissues were soaked in molecular grade

H2O prior to DNA extraction, to remove trace ethanol. DNA was eluted in 100 l elution buffer and quantified using a NanoDrop-1000 (Thermo Fisher Scientific). WSSV-infected and specific pathogen free (SPF) shrimp tissue samples were obtained from the OIE reference laboratory in Arizona and DNA was extracted as above. These were used as positive and negative control material for WSSV in subsequent molecular diagnostic assays. To ensure that amplifiable DNA was present in all extracted samples, crustacean genomic DNAs were assessed using the universal small subunit ribosomal RNA (SSU-rRNA) gene primers 16S-A (5‟-AACCTGGTTGATCCTGCCAGT-3‟) and 16S-B (5‟- GATCCTTCTGCAGGTTCACCTAC-3‟) (Medlin et al., 1988) with an expected amplification product of approximately 1800 bp. Each 20 l reaction contained 1x Green GoTaq Flexi Buffer (Promega), 1.5 mM MgCl2, 0.25 mM of each dNTP, 1 M of each primer, 1 unit Taq polymerase, and 1 l genomic DNA (20-50 ng total). Amplifications were performed with an initial denaturation temperature of 94 C for 4 min, followed by 35 cycles at 94 C for 30 s, 45 C for 30 s, 65 C for 2 min, with a final elongation step at 65 C for 5 min. Following amplification, 5 l of each PCR product were analysed by agarose gel electrophoresis (1.5 % w/v), stained with ethidium bromide, and viewed under a UV light source. Images were captured with a Gel Doc 2000 (Bio Rad) imaging system.

WSSV-infection status of tissues was confirmed using the nested PCR assay of Lo et al. (1996) with minor modifications (Ms. Bonnie Poulos, University of Arizona, personal communication). First, a product of 1447 bp was amplified using the primer pair 146F1 (5‟-ACTACTAACTTCAGCCTATCTAG-3‟) and 146R1 (5‟- TAATGCGGGTGTAATGTTCTTACGA-3‟), followed by an approximate 941 bp product in the nested reaction using primer pair 146F2 (5‟-GTAACTCCCCCTTCCATCTCCA 3‟) and 146R2 (5‟TACGGCAGCTGCTGCACCT-TGT-3‟). For the first round of amplification (primer pair 146F1/146R1)

each 25 l PCR reaction contained the following: 10 mM Tris-HCl (pH 8.4), 50 mM KCl, 2 mM MgCl2, 200 M of each dNTP, 0.31 M of each primer, 2.5 units Taq polymerase, and 1 l genomic DNA (20-50 ng total). Amplifications were performed with an initial denaturation temperature of 94C for 2 min, followed by 30 cycles at 94C for 30 s, 62C for 30 s, 72C for 30 s, with a final elongation step at 72C for 2 min. Reaction conditions and reagent concentrations were the same for the second round of amplification using the 146F2/146R2 primer pair, however 0.5 l of the first round of amplification was used as a template in place of genomic DNA. Following amplification, 10 l samples of the second round PCR product were analysed by agarose gel electrophoresis as described above. WSSV-challenged tissue samples that were negative by PCR were re-analysed for a second time by the nested WSSV PCR assay to confirm the result. PCR amplification products generated in non-target decapod host species exposed to WSSV via feeding of WSSV-infected shrimp tissues were sequenced for confirmation of their identity. Reactions were analysed on an ABI 3130 Avant Genetic Analyser. The final product was compared to known sequences using Basic Local Alignment Search Tool (BLAST) to determine phylogenetic homology. This is in line with OIE confirmatory diagnostic technique for WSSV (Claydon et al., 2004).

Finally, following PCR confirmation of the presence of amplicons of WSSV in tissues of WSSV-exposed non-target decapods, the infectivity of these tissues (from each test species) was tested via bioassay

EVID4 Evidence Project Final Report (Rev. 06/11) Page 9 of 23 exposure to a known WSSV susceptible species (P. vannamei). Tissues from each test species were homogenised using the aforementioned approach for shrimp. Control innoculum was also prepared for each test species using confirmed WSSV PCR negative samples. Inoculate was diluted 1:20 using sterile saline and filtered prior to intramuscular injection of SPF shrimp (10µl g-1 body weight). Pre-trials utilising inoculate generated from C. maenas revealed that dilution to 1:40 was required to prevent rapid death of shrimp following injection (a condition previously termed „protein shock‟ by Lightner, 1996). Shrimp exposed to confirmed-WSSV PCR positive and negative inoculate were observed for a period of 5 days. A total of 14 tanks, containing 5 shrimp per tank were maintained at a water temperature of 24˚C and observed regularly throughout daylight hours. Dead and terminally moribund animals were removed from each tank and sampled as above. At the end of the challenge period (Day 5), all surviving shrimp were sampled. Pleopods were removed from each shrimp and fixed in 100% ethanol for WSSV detection by PCR (as above). The remaining shrimp carcass was injected with Davidson's seawater fixative and shrimp placed whole into the same fixative for histology (as above).

1.2.2 White Spot Syndrome Virus (WSSV): results

Using the EFSA criteria for susceptibility (see Table 1), replication of the agent, pathology, and topographic location of the pathogen within the host, were demonstrated for all non-model UK host crustacean species via the presence of WSSV virions within intranuclear inclusion bodies in tissues of mesodermal and ectodermal origin (see section on pathogenicity below). Furthermore, viability of the agent (within the non- model host) was confirmed via passage bioassay of infected host tissue to SPF P. vannamei. In this case, the majority of SPF shrimp exposed to WSSV positive inoculate arising from non-model hosts died within 3 days post-injection. All mortalities and all remaining shrimp culled at the end of the challenge period (5 days) were PCR positive for WSSV. All shrimp showed pathognomonic signs of WSD infection via histology according to Lightner, (1996) (data not shown). Despite the universal fulfilment of the EFSA WSD susceptibility criteria by the non-model hosts, there were considerable differences between pathological outcomes in the respective species, particularly when the mode of virus dose delivery is considered (Table 2). All non-model species, apart from C. maenas and A. pallipes fulfilled all four susceptibility criteria via feeding and injection. In the case of C. maenas, replication was not observed in target tissues from animals fed with WSSV, however when tissues from C. maenas formed the basis of the inoculate for bioassay to SPF P. vannamei, characteristic signs of WSD, and mortalities occurred in shrimp. This finding is suggestive of infection in C. maenas with viral loading below that required to cause pathology in target tissues of this species, at least within the time frame of the study. Replication in injected A. pallipes could not be confirmed due to a rapid, non-specific mortality following injection of the inoculate. Replication was demonstrated however following feeding.

Cancer pagurus Homarus Nephrops norvegicus Austropotomobius Pacifastacus Eriocheir sinensis Carcinus maenas gammarus pallipes leniusculus Group Group 3 Group Group 3 Group 2 Group 3 Group 2 Group 3 Group 2 Group 3 Group 2 Group Group 2 Group 3 2 2 3 Gill * *** * *** * *** *** m *** *** *** *** - * Heart - * * ** - ** * m * * *** *** - * Ovary - * - ** - ** * m * * ** ** - - Testis - * - * - * * m * * * * - - Nerve N/A N/A N/A N/A - ** - * ** m * * ** ** Connective Tissue * ** * ** * ** ** m *** *** *** *** - - Cuticular epithelium * ** * *** * *** *** m *** *** *** *** - - Fixed Phagocytes * ** * ** * ** *** m *** *** *** *** - - Haemolymph * ** * * * * ** m ** ** *** *** - - Table 3

Key: * Few infected nuclei present, ** Infected nuclei prevalent throughout tissue, *** Infected nuclei highly prevalent throughout tissue, - Pathology not evident, m Pathology not evident due to mortalities

A summary of pathological observations in WSSV infected non-model hosts is provided in Table 3 (above). In all species, WSSV-associated pathology was most pronounced within the cuticular epithelium of the gills (Fig. 2). WSSV-infected nuclei were enlarged with marginalized chromatin, and often contained a distinct eosinophillic inclusion body. Infected nuclei were also identified within other regions of the cuticular epithelium. Within the heart, the nuclei of spongy connective tissue cells of the epicardium displayed characteristic WSSV inclusions as did nuclei in the region of the sarcolemma surrounding the muscle fibres of the myocardium. Haemocytes and connective tissues, particularly surrounding the haemal vessels and nerves also showed pronounced signs of infection. The germinal epithelium surrounding the oocytes and the connective tissues of the testes also displayed characteristic signs of WSSV infection in some species.

EVID4 Evidence Project Final Report (Rev. 06/11) Page 10 of 23

Fig. 2. WSSV associated pathology within the epithelial cells of the gill of signal crayfish (Pascifastacus leniusculus). A full histopathological atlas of pathologies associated with WSSV infection in UK hosts is given in Bateman et al. (2012a)

The Lymphoid organ (LO), present in members of the Superfamily Penaeoidea (including members of the genus Penaeus) was absent from the non-model hosts considered in this study. The characteristic pathologies of the LO previously associated with WSSV infection in penaeid shrimp, was mimicked by a similar manifestation in fixed phagocytes, particularly within the haemal sinusoids of the hepatopancreas. Fixed phagocytes within the haemal spaces displayed enlarged nuclei containing eosinophillic inclusions and marginated chromatin (Fig. 3). In advanced stages of disease, infected cells appeared to degenerate, with remnant nuclear fragments and cellular debris occurring within the haemal spaces of the hepatpopancreas. Despite such pronounced manifestation of WSSV infection within the fixed phagocytes of the hepatopancreas, the epithelium of the hepatopancreatic tubules per se did not display any obvious signs of infection.

Fig. 3. WSSV associated pathology within the fixed phagocytes within the hepatopancreas of signal crayfish (Pascifastacus leniusculus). A full histopathological atlas of pathologies associated with WSSV infection in UK hosts is given in Bateman et al. (2012a)

Some exceptions to this general pattern were observed. Histological assessment of tissues and organs from WSSV-injected A. pallipes revealed an apparent widespread apoptosis of haemocytes, and a pronounced bacteraemia of the haemolymph in most samples. In these cases, 100% mortality occurred within 48 hours of injection and despite all samples displaying positivity for WSSV at time of death (positive PCR result), it is conceivable that this may have resulted from detection of residual inoculate rather than virus present within infected host cells. In C. maenas, WSSV-associated lesions were observed within the tissues of the WSSV-injected group but at a much lower severity than that seen in the other non-model species. In such cases, very few nuclei containing WSSV-associated inclusions were observed. No pathology was observed in C. maenas fed with WSSV contaminated feed (see Table 3).

All non-model host species were sourced from wild populations in the United Kingdom. Since these hosts were potentially infected with other pathogens at the start of each trial, it is important to consider the effect that these may have had on the outcome of challenge trials. In addition to pathologies associated with infection by WSSV, other conditions recorded at the termination of challenge trials included: Pink Crab Disease (Stentiford et al., 2002) caused by the dinoflagellate Hematodinium sp. in 27% of C. pagurus; parasitic copepod Nicathoë astaci in 50% of H. gammarus; Austropotamobius pallipes Bacilliform Virus (ApBV) (Edgerton et al., 2002) in 60% of A. pallipes; the microsporidian Hepatospora eriocheir (Stentiford et al., 2011) in 51% of E. sinensis. Carcinus maenas were infected by a range of pathogens including the

EVID4 Evidence Project Final Report (Rev. 06/11) Page 11 of 23 digenean Microphallus primas (Saville and Irwin, 2005), the parasitic rhizocephalan Sacculina carcini (Boschma, 1955), bacterial „Milky disease‟ (Eddy et al., 2007), a novel microsporidian infection of the musculature, and Hematodinium perezi (Chatton and Poisson, 1931). In the majority of challenge studies, underlying infections did not cause significant mortalities in control groups. However, an unidentified bacterial infection caused mortality in 12 % of control P. leniusculus. In addition, the WSSV-fed C. maenas group displayed 7% mortality which upon histological examination was apparently due to a pronounced microsporidian infection of the musculature rather than to WSD.

Transmission electron microscopy (TEM) and PCR followed by sequencing are recognised as confirmatory tools for diagnosis of WSSV infection according to the OIE (2009). TEM of gill from each non-model species used in the current study revealed the presence of WSSV virions within hypertrophied nuclei of infected cells. Nuclei were enlarged with marginalised chromatin and contained virions at varying stages of development (Fig. 4). Fully formed virions were ovoid to elliptical in shape and contained an electron dense nucleocapsid surrounded by a trilaminar envelope. The dimensions of virions varied according to stage of development but measurements of fully formed virions were consistent with those previously described for WSSV. During patent WSD in E. sinensis, paracrystalline arrays of WSSV virions were observed at the periphery of infected nuclei. Un-enveloped nucelocapsid material was present within the nuclei of some hosts this displayed the characteristic stacked sub-units of the WSSV genome. Interestingly, viral particles present within infected nuclei of C. maenas tissues were morphologically distinct from characteristic WSSV virions, although un-enveloped nucleocapsid material also appeared identical to that observed in WSSV. The nucleocapsid within intact viral particles in various stages of development in C. maenas appeared curved or „u‟ shaped within the viral envelope.

Fig. 4. Transmission electron microscopy of WSSV infecting crustacean hosts. Virions form arrays within the host nucleus (in this case in the European edible crab Cancer pagurus) and in isolation are shown to be large, enveloped and elliptical. Further ultrastructural descriptions of WSSV in UK hosts are provided in Bateman et al. (2012a).

PCR analysis of gill tissues from the WSSV-injected sample group of all species revealed universal positivity for WSSV. PCR analysis of gill tissues from WSSV-fed groups of the different non-model host species revealed varying proportions of WSSV positive samples as follows: 33% C. pagurus, 20% C. maenas 17% H. gammarus, 65% N. norvegicus, 10% E. sinensis, 40% A. pallipes and 5% P. leniusculus. For confirmation of identity, a single WSSV PCR-positive sample from each of test species was sequenced. All sequenced samples showed between 99.6% and 99.8% similarity to the WSSV isolate utilised for challenge (OIE isolate of WSSV UAZ 00-173B).

Overall, these results confirm that the UK crustacean host species tested in this study were considered susceptible to infection with WSSV and that the virus is able to replicate and remain virulent within these species via onward passage to a known susceptible host, Penaeus vannamei. However, relative susceptibility of the different hosts differed significantly and will be discussed further below.

1.2.3 White Spot Syndrome Virus (WSSV): discussion and conclusions

1.2.4 Fulfilling the criteria for susceptibility Studies carried out within FC1186 have demonstrated universal susceptibility to WSSV infection in a range of European decapod crustaceans, exposed to WSSV via a natural route (feeding). Furthermore, we show that WSSV can replicate and cause disease within European crustacean species at ambient water temperatures encountered in European waterways. Despite universal susceptibility, the manifestation of disease associated with prior infection with WSSV differs considerably between species; freshwater crayfish undergoing more rapid mortality and development of pathognomonic signs than most of the marine

EVID4 Evidence Project Final Report (Rev. 06/11) Page 12 of 23 species tested. Despite becoming infected with WSSV following exposure, the European shore crab (Carcinus maenas) appeared most recalcitrant to the development of disease and may be considered as an asymptomatic carrier under those conditions utilised in the current trials. Stentiford et al. (2009) list all of the species which have been shown by experimental and natural exposures to be susceptible to WSD according to EFSA guidelines. In this review, susceptibility criteria were partially fulfilled for several European species. Extension of such work in the current study has shown that WSSV can replicate, cause pathology and that the virus remains viable (by bioassay), thereby fulfilling all four EFSA categories for susceptibility. In summary, we provide definite evidence for susceptibility to WSSV infection in H. gammarus, C. pagurus, C. maenas, N. norvegicus, E. sinensis, P. leniusculus and A. pallipes. This is the first description of susceptibility to WSSV infection in H. gammarus, N. norvegicus, E. sinensis and A. pallipes.

Pathognomonic signs of WSSV infection in non-model crustacean hosts Studies with penaeid shrimp have shown that WSSV targets crustacean tissues of ectodermal and endodermal origin. It is most commonly detected in the cuticular epithelium, particularly associated with the gill and other appendages, or in the epithelial layer surrounding the stomach. Despite the wide susceptible host range to WSSV, few studies have focussed on the pathognomonic signs of infection (and disease) in these species. In the current study, no definitive external signs (such as white spots) were associated with infection or disease in non-model hosts. WSSV infection and WSD could however be detected using histology in a manner similar to that previously described for penaeid shrimp. Characteristic eosinophillic inclusion bodies were observed within the hypertrophic nuclei of WSSV infected hosts cells. In penaeids, such eosinophillic bodies are usually observed in early stage WSSV infection, these staining more basophilic as disease progresses. Although this indicates that the pathology within individual cells of WSSV-infected non-model hosts were at a relatively early stage, longer term exposure trials may have led to a higher frequency of basophilic types as observed in penaeid hosts. Fed animals displayed fewer WSSV-infected nuclei per tissue section, this most likely due to the lower viral loading of single feed dose compared to those directly injected with viral inoculate.

WSSV inclusions were most evident in epithelial cells of the gills and other regions of sub-cuticulum, within haemocytes, connective tissue cells, fixed phagocytes, heart myofibrils and spongy pericardial cells. The absence of a lymphoid organ in hosts other than members of the Superfamily Penaeoidea (including the genus Penaeus) prevent this organ being used as a sentinel for viral infection in non-model hosts. However, characteristic pathognomonic signs comparative to those observed within penaeids during WSD, were seen in the fixed phagocytes of the hepatopancreas of non-model hosts, suggesting that this system is progressively degraded during pathogenesis of WSSV infection in non-model hosts. It is likely therefore that the fixed phagocyte system provides a comparative target (to the lymphoid organ) for WSSV in non- model crustaceans.

Infection severity within specific tissues varied considerably from species to species though in the majority of cases, the tissues of the gill provided a reliable means to diagnose infection via histology. Although considerable variation in pathology was observed between fed and injected groups for some species, in the freshwater crayfish (P. leniusculus, A. pallipes) and Chinese mitten crab (E. sinensis) significant pathology was observed, even in fed animals, during the short duration of exposure trials. These results are suggestive of more rapid pathogenesis of WSSV infection in these species. In some instances, WSSV- infected host nuclei were evident within the supporting cells and other connective tissue cells surrounding the oocytes and spermatozoa. Whether this reflects an ability for vertical transmission in non-model crustacean hosts remains to be demonstrated.

Whilst PCR (and sequencing) was utilised to confirm the presence of WSSV in non-model crustacean hosts, it should be noted that PCR alone is not adequate to confirm host susceptibility per se. Since the technique simply detects the presence or absence of elements of the viral genome, its sole use cannot be applied to unequivocally demonstrate host cell infection by a given pathogen. For this reason, PCR data is not utilised to assess host susceptibility according to EFSA (see Stentiford et al., 2009).

1.2.5 The concept of relative susceptibility

Whilst adding to the remarkable list of species susceptible to WSSV infection (Stentiford et al., 2009), we have highlighted a significant variation in pathogenesis in the different non-model species investigated in this study. By considering this data alongside the mortality rates observed during the period of specific trials we have defined three broad categories of relative susceptibility to WSSV infection and disease in crustacean hosts (Table 4). In such a way, the concept of „susceptibility‟ to infection and disease in crustaceans can be considered both at the species level, but also at the level of the individual, with factors such as exposure dose, moult status, physiological condition and the presence of pre-existing pathogen infections disease likely playing a part in progression of infection to disease, and the propensity for mortality to occur. We have shown that some species such as the freshwater crayfish species P. leniusculus and A.

EVID4 Evidence Project Final Report (Rev. 06/11) Page 13 of 23 pallipes display characteristic pathognomonic signs and undergo rapid mortality following single-dose feeding of WSSV-contaminated shrimp carcasses. In this context, they are classified alongside penaeid shrimp as ‟highly susceptible‟ to WSSV infection and its disease, WSD. In contrast, C. maenas does not display widespread pathognomonic signs, nor does it undergo significant mortalities when exposed to WSSV, either via feeding or direct injection. This species is therefore considered to display „low susceptibility‟ to WSD (despite being able to be infected with WSSV via both feeding and direct injection). Low susceptible hosts, which presumably also harbour low viral titres, can however passage sufficient doses to cause disease and mortality in highly susceptible hosts (such as the SPF P. vannamei utilised here). In this context, they are considered as asymptomatic carriers for WSSV. Further work presented in section 2 (below) investigated the potential for initiation of disease in low susceptible hosts (carriers) during ambient stressor events.

Table 4

Mortality Pathology Species Type 1 - High High mortality in both Classic white spot pathology Penaeid shrimp injected and fed obvious in tissues from both Austropotamobius pallipes exposures fed and injected exposures Pacifastacus leniusculus Eriocheir sinensis

Type 2 - Medium High mortality in injected Classic white spot pathology Homarus gammarus exposure, little or no obvious in tissues from Nephrops norvegicus mortality in fed exposure injected exposure. Little or no Cancer pagurus pathology evident in fed exposure

Type 3 - Low Low level mortality in Little or no pathology evident Carcinus maenas both injected and fed in either injected or fed exposures exposures

1.2.6 The basis of differential susceptibility to WSSV One species, Carcinus maenas, clearly showed a lower susceptibility to WSSV when compared to other European decapod species. WSSV, classified within a single genus Whispovirus in the family Nimaviridae is currently the only member of this genus. The literature also tentatively lists so-called B virus from Carcinus maenas, B2 virus and λ (tau) virus from Carcinus mediterraneus, and Baculo-A and Baculo-B viruses from Callinectes sapidus as potential members of the genus. The B, B2, and Baculo-B viruses are extremely similar in terms of size, shape and morphogenesis to that reported from WSSV; in fact these viruses appear morphologically indistinguishable and it has been suggested that they may in fact be ancestral forms of WSSV. Anecdotal evidence suggests that during initial outbreaks of WSSV in Asian penaeid farms, broodstock shrimp were fed with carcasses of portunid crabs (Prof. Grace Lo, National Taiwan University, Pers. Comm.). It is conceivable therefore that the virus now plaguing the global penaeid shrimp farming industry may have its origins in crabs. As such, these ancestral hosts may well possess genome-based mechanisms to deal with viruses such as WSSV. In this context, historical exposure of Carcinus spp. to B/B2 virus, which is similar if not identical to WSSV at least at morphological level, may have inferred inherited recalcitrance to WSSV. The research team responsible for delivering FC1186 have proposed further work rediscover B/B2 virus and to perform comparative phylogenetic analysis against WSSV from penaeid hosts.

1.2.7 Predicting consequence of WSD for UK crustacean stocks The European crustacean industry centres on commodity imports (e.g. frozen tropical shrimp) and on wild fisheries, with limited aquaculture production of crustaceans (Stentiford et al., 2010). Wild fisheries for marine crustaceans are considered key resources in the European area with total fishery production of crustaceans from European waters totalling 120,000Mt in 2008 (www.fao.org/figis). Import of commodity products has been highlighted as a possible route of introduction of WSSV to new locations. In some regions, shrimp imported for human consumption are diverted for use as angling baits; this identified as a significant risk factor for the introduction of viral crustacean pathogens. In FC1186 we have demonstrated susceptibility of several European decapods to WSSV-infection following a single meal of contaminated feed and further, that the virus remains viable for passage to other susceptible hosts. Significantly, this was even shown to occur when the donor host displayed show no characteristic signs of infection or disease. Further work is needed to assess the full risk of imported commodity product to European crustacean species, particularly due to anecdotal information relating to the use or raw shrimp commodity as angling bait in UK freshwater and marine environments. Pilot work relating to this is presented in Section 3 (below)

EVID4 Evidence Project Final Report (Rev. 06/11) Page 14 of 23 Although we have shown that European species are susceptible to WSD it is not known whether infection would lead to transmission, establishment, disease and elevated mortality in exposed populations. Previous studies have suggested that severe infections (with WSSV) are rarely seen in the wild and suggest that this may be due to limited monitoring of such populations, the elevated vulnerability of diseased individuals to predation, the swift progression of disease, or in fact to the rarity of the event occurring. Similar studies in the USA (on blue crabs Callinectes sapidus) along report reasonably high level prevalence with WSSV but that these populations do not appear to be symptomatic for WSD (suggesting they instead act as reservoir hosts). It is also important to note that WSSV has been shown to be carried in some farmed shrimp populations at low intensities in low-stress culture conditions without mortality events occurring. It is possible therefore that even highly susceptible species may be able to tolerate infection while conditions are favourable, but may succumb to the disease when sub-optimal conditions occur. Disease outbreaks on shrimp farms are known to be induced by stressors such as rapid change in salinity and drop in temperature. How the latent disease or „carrier‟ status will alter during stressful events such as moulting, reproduction and disease with non-penaeid species is unclear and requires further studies. Longer term studies than those presented in this first section were deemed necessary to address the issue of whether low-level infections in wild (non-model) crustaceans would persist as such for extended periods or whether stressors cause progression of infection to disease at the individual and population levels (these trials are presented in Section 2 below).

1.2.8 Final comments on WSSV susceptibility

Although most commonly associated with disease in tropical shrimp, one of the first reports of WSSV described mortalities of Marsupenaeus japonicus in water temperatures of 19˚C. This water temperature is experienced within the Southern European region over several seasons and even in Northern Europe (e.g. UK) during summer months. Although temperature has been shown to affect the replication of WSSV and the expression of WSD in penaeid shrimp and crayfish (both undergoing reduced mortality rates and lower viral loading in both hypothermic and hyperthermic conditions), disease develops rapidly when infected animals are returned to optimal conditions for the pathogen. These findings suggest that susceptible hosts exposed to WSSV at sub-optimal temperatures for viral replication may not develop disease but that disease (and mortality) may occur when conditions become more favourable for viral replication. During this time however, they may serve as a reservoir to spread the virus to other naive hosts. Transmission from asymptomatic carrier status hosts to other hosts of the same species or disease susceptibility (e.g. low, medium, high) will be required to assess the true risk of WSSV to wild decapod crustacean populations following introduction of the pathogen. This data is critical in assessing the consequence of WSSV infection to European crustacean populations. Due to difficulties in obtaining and maintaining lower crustaceans (e.g. amphipods) during the current study, challenge protocols were not successfully developed. This would be a fruitful area for onward research since these animals form important components of aquatic ecosystems.

1.3 Taura Syndrome Virus (TSV) and Yellowhead Virus (YHV): exposure trials

The review by Stentiford et al. (2009) revealed that TS and YHD (associated with infections by TSV and YHV, respectively) were mainly considered as penaeid-specific pathogens, infecting farmed shrimp from tropical regions. However, in light of limited availability of data concerning susceptibility of non-penaeid hosts, particularly those from temperate water habitats, to these viruses, we carried out a series of challenge trials using both invasive (injection) and natural (feeding) routes of infection, in line with those approaches outlined above for WSSV.

1.3.1. Signal crayfish (Pacifastacus leniusculus) challenge: YHV and TSV

Challenge trials were conducted using direct injection of TSV or YHV into signal crayfish (P. leniusculus) using inoculate prepared from confirmed TSV/YHV infected penaeid shrimps (generated as part of FC1186). Animals were held at 20oC for the duration of the experiment (10 days) following which samples were obtained for histology, TEM and molecular diagnostics as above. For the YHV experimental trial, mortality was not elevated above negative control animals, no characteristic pathologies were observed via histology, and all samples were confirmed negative for YHV via PCR. For the TSV experimental trial, mortality was not elevated above negative control animals, no characteristic pathologies were observed via histology, but several samples were confirmed positive for TSV via PCR. Material from these PCR positive animals is currently being passaged via bioassay back to known susceptible hosts (P. vannamei) to confirm susceptibility of P. leniusculus to TSV. These results will be reported to Defra as soon as available.

1.3.2. European shore crab (Carcinus maenas) and shrimp (Palaemon elegans) challenge: TSV

A further study investigated the susceptibility to TSV of two European crustaceans, the shore crab Carcinus maenas and the rock pool shrimp Palaemon elegans. Ten shore crabs and 40 rock pool shrimp were fed with TSV-infected tissue of Penaeus (Litopenaeus) vannamei and 10 shore crabs were injected with a TSV

EVID4 Evidence Project Final Report (Rev. 06/11) Page 15 of 23 viral homogenate. Five shore crabs and 40 rock pool shrimp fed with uninfected P. vannamei carcasses and 5 shore crabs injected with saline solution served as negative controls. Animals were held at 18oC. The haemolymph of crabs and body tissues of shrimp sampled at 7 and 20 days post-challenge was tested for TSV by RT-PCR. One of the shore crabs injected with TSV tested positive at day 7 post-challenge, while one TSV-fed and six TSV-injected shore crabs tested positive on day 20 post-challenge. No TSV was detected in challenged rock pool shrimp. No histopathological abnormalities were found in TSV-infected crabs sampled at 20 days post-challenge. An infectivity assay was performed to determine if crabs that tested positive for TSV by RT-PCR could transmit TSV to P. vannamei. A tissue homogenate from TSV- infected crabs was injected into five P. vannamei. Four of these shrimp exhibited signs of TSV infection and 4 died 7 days post challenge. TSV was detected in the tissues of challenged P. vannamei by RT-PCR. The results of this study suggest that similar to P. leniusculus (1.3.1 above), Carcinus maenas may act as an asymptomatic carrier for TSV at water temperatures that occur within European waters. Longer term trials are required to investigate potential for formation of disease in this species.

1.3.3 European lobster (Homarus gammarus) challenge: TSV and YHV

This trial utilised hatchery reared juvenile lobsters in a similar manner to that described for WSSV (section 1, above). For the TSV and YHV challenge trial, water temperature was held constant at 22oC. Lobsters in Tank 1 (n = 7) were fed a single ration (0.05g) of TSV-infected shrimp tissue on Day 0. Lobsters in Tank 2 (n = 7) were fed a single ration (0.05g) of YHV-infected shrimp tissue on Day 0. Lobsters in Tank 3 (n = 7) were fed a single ration (0.05g) of uninfected shrimp tissue on Day 0. Thereafter, lobsters in all tanks were fed on 3 mm Royal Oyster pellets at a ration of approximately 3-4 % bodyweight/day. Tanks were observed regularly throughout daylight hours for the following 10 days. Claw samples from all dead, terminally morbid and live (at the end of the 10 day trial period) animals were prepared for PCR. Remaining carcasses were prepared for histology and selectively, for electron microscopy. No mortalities were observed in either TSV- fed or YHV-fed lobsters, or in control animals over the 7 day period of the trial. Control lobsters and those fed TSV-infected and YHV-infected shrimp tissues and sacrificed at the end of the experimental period did not exhibit histopathological alterations in any tissues or organs that has previously been associated with infection by these viruses. However, PCR revealed that one of the lobsters was positive for TSV nucleic acid at the end of the 10 day trial. A few animals also showed suspect lesions, consistent with that known for penaeid shrimp, in the intestinal epithelium. No animals were positive for YHV via PCR.

Given the significant finding of PCR positivity for TSV in lobsters exposed via the natural route of feeding, and the contrast to data reviewed by Stentiford et al. (2009) for TSV susceptibility, the trial was repeated using juvenile lobsters obtained from the commercial hatchery. In the repeat trial (all major conditions consistent with those described above), PCR positivity for TSV was also detected in pooled lobster samples at the end of the 10 day exposure period. Furthermore, pathological alterations somewhat consistent with those observed in TSV-infected penaeid shrimp were observed in TSV-fed lobsters (Fig. 5).

Fig. 5. Lesions observed within connective tissue and epidermal cells of TSV-exposed H. gammarus. Pyknotic nuclei and strongly-eosinphilic cytoplasm is consistent with the pathology of TSV infection in penaeid shrimp.

1.3.4. Susceptibility of European crustacean species to TSV and YHV: conclusions

The results of these challenge trials suggest that the signal crayfish (P. leniusculus), European shore crab (C. maenas) and European lobster (H. gammarus), can obtain infection with TSV when challenged by injection or feeding, and that TSV may survive in C. maenas for at least 20 days post-infection. In contrast, no experimentally challenged rock pool shrimp (P. elegans) tested positive for TSV by RT-PCR. These findings support recent data which reports some potential for several Asian crab species as „susceptible‟ to infection with TSV, and that crabs may be permissive to TSV replication. Although no pathological abnormalities were evident in TSV-infected shore crabs, including crabs that tested positive for TSV by RT- PCR, some TS-like lesions were observed in juvenile lobsters via feeding on infected shrimp carcasses.

Interestingly, in the shore crab study (which sampled at 7 and 20 days), crabs which were TSV negative at

EVID4 Evidence Project Final Report (Rev. 06/11) Page 16 of 23 7 days became positive after 20 days post-infection, suggesting that replication and subsequent circulation of TSV occurred within challenged animals. Further trials using quantitative PCR (qRT-PCR) could now be used to demonstrate relative viral titre but these studies were out-with the scope of FC1186. Regardless of viral titre, confirmed TSV-infected crab tissue, when passaged back to the susceptible model host (P. vannamei) caused TS in these shrimp, confirming the infection in crabs and demonstrating viability in the virus following crab infection. The lobster studies (repeated) provide further confirmation that TSV is able to infect temperate water species via natural routes of exposure (feeding) and further, that TSV retains infectivity for significant periods within these hosts.

In summary, we have reported the results of challenge experiments which suggest that the certain European crustacean species appear susceptible to infection with TSV according to EFSA criteria as detailed in the review by Stentiford et al. (2009). This has important implications for the health management of European crustaceans both in the wild and in captivity. This shore crab is common in several temperate areas including some that are important both for penaeid fisheries and aquaculture. European lobsters are an important fisheries resource (e.g. in the UK) and are also traded live to international markets. Further research is needed to assess the sensitivity of native penaeid species and other crustaceans to TSV, and the putative role of the crayfish, shore crab and lobster as carriers of this virus.

2 Defining the effects of stress and ambient conditions on WSSV pathogenesis in UK crustacean hosts (Objectives 3 and 5).

2.1 Effects of temperature on WSSV pathogenicity (Carcinus maenas)

Since an interesting feature of the studies on the pathology of WSSV infection in UK marine hosts has been the demonstration of apparently reduced replication rate in shore crabs (C. maenas), and further that this species may be targeted by the UK as a survey organism for demonstration of freedom from White Spot Disease (WSD) caused by WSSV (a requisite for Member States wishing to declare freedom according to EC Directive 2006/88), we have carried out further detailed challenge trials using this species during the period of this report (Objectives 3 and 5). Specifically, we have investigated the potential for transition from „carrier status‟ to „diseased‟ in this apparently low-susceptible host. In this trial, we retained two WSSV-ve control groups (20oC, and 20oC rising to 26oC over three weeks) and two WSSV+ve groups (20oC, and 20oC rising to 26oC over three weeks) for a period of 6 weeks. The challenge was designed to emulate a typical summer period in the UK whereby the micro-climate of shoreline habitats (e.g. rockpools) may experience elevated temperatures for relatively extended periods. By utilising a common shoreline species (C. maenas) that happens to demonstrate relatively low susceptibility to WSD we can directly assess the potential for UK temperatures to limit disease progression and mortality in this type of low-susceptible species. Whilst these data are currently being analysed and will likely form a further peer-reviewed manuscript submission, preliminary analysis reveals that at least for this species, elevated summer temperatures (i.e. up to 26oC) do not necessarily cause an increased mortality in WSSV exposed hosts. This is summarised in Fig. 6.

Fig. 6. Mortality of Carcinus maenas injected with WSSV inoculums or sham controls, both at constant 20oC or 20oC rising to 26oC. The application of a temperature stressor did not affect the pathogenesis of WSSV in this species.

EVID4 Evidence Project Final Report (Rev. 06/11) Page 17 of 23

In these relatively extended tank studies, while it is demonstrated that mortality is increased over control levels in WSSV-exposed C. maenas (green and purple lines), these levels of mortality (~30% in 6 weeks) are considerably lower that would be observed in other species so far investigated (see above). Furthermore, increasing water temperature from 20oC to 22oC (day 7), 22oC to 24oC (day 14) and 24oC to 26oC (day 21) (purple line) did not lead to elevated mortality compared to those animals exposed to WSSV and retained at 20oC for the duration of the study (green line). These data suggest that a. Ambient water temperature of 20oC is sufficient for replication of WSSV, and b. temperature is not the limiting factor for WSSV viral replication in this host species. The basis for reduced pathogenesis of WSSV in C. maenas is now the target of further study but likely indicates some hereditary capacity for relative resistance in this (and likely other hosts). Other species (such as N. Norvegicus, H. Gammarus and the freshwater crayfish) do not appear to possess such hereditary capacity, with significant mortality occurring in very short time scales (e.g. 5 days) after WSSV exposure. Natural genetic resistance markers are an obvious target of interest in hosts such as C. maenas and may relate to their exposure (in evolutionary terms) to similar large DNA viruses.

2.2 Low temperature exposure to WSSV in lobsters (Homarus gammarus)

In order to assess the effect of temperature on the susceptibility and pathogenesis of WSSV in lobsters, we exposed 120 juvenile lobsters to confirmed-WSSV-contaminated shrimp feed, and 120 juvenile lobsters to WSSV-negative control shrimp feed. Lobsters were subsequently maintained at a range of ambient water temperatures typical within the UK (8oC, 10oC, 13oC, 16oC, 20oC). Animals were retained for 10 days or until mortalities occurred. In summary, infection (confirmed using histology in the first instance) was possible at all temperatures, though progression to disease was maximal at the highest temperatures tested. These results suggest that lobsters may obtain WSSV infection, even when exposed at low (winter) temperatures. Progression to disease status however appears to be limited by ambient conditions, confirming results presented in 1.2.2 (above) and reported in Bateman et al. (2012a).

2.3 Longer term studies of WSSV exposure (Carcinus maenas)

Studies designed to assess susceptibility of a potential host to a particular pathogen are generally short term, and assess a range of parameters which either confirm or refute susceptibility in that host (Stentiford et al. 2009). However, throughout the course of the current project, the consequence of susceptibility in those host species which appeared to show recalcitrance to disease was also assessed. Here, 42 C. maenas were fed with once with WSSV-contaminated feed and retained within flow-through aquaria at 20oC, for a period of 3 months. A further 40 crabs were fed once with WSSV-negative control feed and retained in similar conditions. Mortalities were recorded and removed/processed for histology, TEM and molecular diagnostics throughout the study. All other animals were processed for the same techniques at the end of the 3 month period. Very few mortalities occurred within each group (5 in control, 6 in WSSV tank) throughout the 3 month period. These were not associated with WSSV positivity (via PCR and histology) in the latter. However, at the end of the trial (3 months), two crabs from the WSSV-fed group were positive for WSSV via PCR. Tissues from these crabs (plus selected negative examples) are currently being bioassayed back to susceptible shrimp hosts to confirm infection. These results provide evidence that WSSV can be retained in the tissues of low susceptible hosts such as C. maenas for extended time periods without progressing to disease and further, that infection can occur following a single feed on WSSV- infected shrimp feeds. The result also supports the potential for C. maenas to act as an asymptomatic carried of WSSV and the low likelihood that infection will progress to disease in this species. However, bioassay from such carrier hosts back to highly susceptible hosts provide evidence that carriers may play an important role in maintaining an infection source within the environment should an outbreak occur. Further, these hosts may become the source for re-infection/mortality in highly susceptible hosts at some point in the future.

3 Towards informing risk assessment for establishment of WSD, TS and YHD in UK crustacean hosts: a study of supermarket shrimp commodity (Objective 5)

3.1 Does supermarket-derived commodity shrimp contain viable WSSV?

EC Directive 2006/88 requires health certification for import of commodity products unless these products are destined for further processing, packaged in „retail sale‟ packages, and labelled in accordance with EC Regulation 853/2004. Therefore, products (live or frozen) imported directly for human consumption are not covered by the Directive and do not need to originate from areas designated free from listed pathogens, even when imported to confirmed „disease free‟ Member States. Previous studies in the USA have demonstrated that frozen commodity shrimp imported for human consumption tested positive for WSSV by PCR. Further, transmission trials using such commodity shrimp have been used to demonstrate viability of the virus in frozen products. Oidtmann & Stentiford (2011) highlight the risk of these commodity products to

EVID4 Evidence Project Final Report (Rev. 06/11) Page 18 of 23 naive crustacean populations by stating “...if such shrimp were introduced into a country free from the pathogen, and crustaceans in the receiving country were exposed to infected tissues per os, there is a considerable risk that such exposed crustaceans may become infected and the infection established in domestic populations of crustaceans”.

Under FC1186, imported fresh and frozen shrimp products obtained from local supermarkets and from a large London fish market were screened for the presence of WSSV nucleic acid. Imported commodity was also tested for potential to act as a source for transmission of WSSV to a known WSSV-susceptible host (P. vannamei) and an important species in the European crustacean fishery, the European lobster (Homarus gammarus). Juvenile H. gammarus obtained from a commercial hatchery in the United Kingdom were exposed to known high-dose (high WSSV viral load) feed at two temperatures to determine initial susceptibility of this species. In addition, juvenile H. gammarus were fed high–dose WSSV infected P. vannamei carcasses and low-dose (low WSSV viral load) supermarket-derived commodity product. Viral loading in feed was assessed prior to feeding using quantitative PCR (QPCR). The susceptibility of juvenile lobsters to WSSV was assessed using histopathology, electron microscopy and nested PCR assays specific for WSSV. The full results from this trial are presented in the peer-reviewed publication of Bateman et al. (2012b).

3.2. Approach

Frozen uncooked shrimp from various global production regions were purchased from supermarkets and from a large fish market in the United Kingdom. Products were tested for the presence of WSSV using a nested PCR assay recommended by the OIE (OIE, 2009). Products displaying positivity via PCR were utilised for production of inoculates and feeds for subsequent passage trials. All passage trials were conducted within the biosecure exotic diseases facility at the Cefas Weymouth laboratory and utilised local, filtered and UV treated seawater. Day length was set at 14 h/day, night was at 10 h with a 30 min fade to simulate dusk and dawn. Temperature was regulated according to the experimental conditions required for Penaeus vannamei and for Homarus gammarus as appropriate.

Commodity shrimp from Ecuador and Vietnam which had been confirmed positive for WSSV via nested PCR were macerated using a sterile razor blade prior to homogenisation in sterile saline (4 ml of saline per gram of minced tissue) using a blender until tissues were liquefied. The homogenate was centrifuged at 5,000 x g for 20 minutes at 4°C to pellet solid debris prior to the supernatant being diluted 1:20 with sterile saline and filtered (0.45 µm) to form the innoculum. Individual specific pathogen free (SPF) L. vannamei, (approximately 5g in weight) obtained from the Centre for Sustainable Aquaculture Research (CSAR) at the University of Swansea, United Kingdom, were inoculated via intramuscular injection of the diluted viral homogenates at a rate of 10 l g1 shrimp weight. Water temperature was held constant at 24˚C. Shrimp were monitored throughout the day for five days, dead and moribund shrimp were removed from the experimental tanks; pleopods were fixed in ethanol for molecular analysis, gills were taken for transmission electron microscopy and the carcass was fixed whole in Davidson‟s seawater fixative for histopathological confirmation of WSD.

For preparation of high-dose feeds, viral inoculates of WSSV were obtained from the OIE reference laboratory at the University of Arizona, USA. The OIE isolate of WSSV (UAZ 00-173B) was generated in L. vannamei from an original outbreak in Fenneropeneaus chinensis (Holthuis, 1980) in China in 1995. Subsequent passages of this isolate into naïve L. vannamei held at the Cefas Weymouth laboratory have demonstrated continued pronounced virulence of this isolate (data not reported here). High-dose WSSV infected shrimp carcasses were prepared by direct injection of the University of Arizona isolate into SPF L. vannamei as detailed above. The viral loading in high-dose feeds was assessed using QPCR. Abdominal tissues from L. vannamei that were confirmed positive for WSSV via histology and nested PCR were macerated into approximately 2-3 mm blocks using sterile razor blade. For preparation of low-dose feeds, individual supermarket-derived shrimp (abdominal section) originating from Ecuador, Vietnam and Honduras, and confirmed positive for WSSV via nested PCR were macerated. The viral loading in low-dose feeds was assessed using QPCR.

Juvenile European lobsters (Homarus gammarus) were obtained from the a commercial hatchery. Lobsters were at moult stage 4 and approximately 2 months of age. To prevent conflict, juvenile lobsters were housed individually in custom-made „Orkney pots‟ that were suspended in the upper water column of each 30 litre experimental tank. The pots contained a perforated base to allow for water circulation.

In Trial 1, water temperature was held constant at 15oC. Lobsters in Tank 1 (n = 20) received a single ration (~ 0.05 g) of high dose WSSV-infected shrimp tissue on Day 0 and a further ration on Day 7. Lobsters in Tank 2 (n = 20) received a single ration (~ 0.05 g) of uninfected shrimp tissue on Day 0 and another on Day 7. Between these times lobsters were fed on 3 mm Royal Oyster pellets at a ration of approximately 3-4% bodyweight/day for 10 days. In Trial 2, water temperature was held constant at 22oC. Lobsters in Tank 1 (n

EVID4 Evidence Project Final Report (Rev. 06/11) Page 19 of 23 = 20) received a single ration (0.05 g) of high dose WSSV-infected shrimp tissue on Day 0 and a further ration on Day 7. Lobsters in Tank 2 (n = 20) received a single ration (0.05 g) of uninfected shrimp tissue on Day 0 and another on Day 7. Thereafter, lobsters in both tanks were fed on 3 mm Royal Oyster pellets at a ration of approximately 3-4% bodyweight/day for 10 days. In Trial 3, water temperature was held constant at 20˚C. On day 0 lobsters in all tanks (n=20 for each tank) received a single ration of feed (0.05 g). Tank 1 received high-dose WSSV-infected shrimp tissue, Tank 2 received uninfected shrimp tissue, Tank 3 received low-dose commodity shrimp originating from Ecuador, Tank 4 received low-dose commodity shrimp originating from Vietnam and Tank 5 received low-dose commodity shrimp originating from Honduras. Thereafter, lobsters in all tanks were fed on 3 mm Royal Oyster pellets at a ration of approximately 3-4% bodyweight/day for 12 days.

In all trials, tanks were observed regularly throughout daylight hours. Dead and terminally morbid animals were removed from each tank. Cheliped samples from all dead, terminally morbid and live (at the end of the trial period) animals were fixed in 100% ethanol for PCR. Remaining carcasses were prepared for histology and selectively, for transmission electron microscopy (TEM) and for molecular diagnostics in a similar manner to that presented above (Section 1). Specific commodity shrimp demonstrated to be PCR positive for WSSV using the nested assay were further analysed using a QPCR assay to determine relative viral load of WSSV.

3.3 WSSV in supermarket commodity shrimp

The prevalence of WSSV in commodity shrimp imported into the UK for human consumption ranged from 0% to 100%. Sixty six percent (4/6) of the batches of shrimp purchased from supermarkets tested positive for WSSV, within bag prevalence ranging from 0% to 100%. Ten percent (1/10) of the shrimp samples purchased from the London fish market were also positive for WSSV. Bioassay shrimp injected with homogenised WSSV positive commodity shrimp from either Ecuador or Vietnam experienced 100% and 40% mortality respectively, within 3 days post injection. Nested PCR analysis indicated that all animals injected with commodity inoculate from Ecuador were positive for WSSV, with four of these animals displaying infection detectable in the first round of PCR, indicating a pronounced disease status. Shrimp injected with commodity inoculate from Vietnam were similarly all positive for WSSV but only weak bands were present in the second round of PCR, suggestive of a lower level infection. Histological examination of these shrimp demonstrated the characteristic pathology associated with WSSV infection. Shrimp injected with commodity inoculate from Ecuador displayed signs of advanced WSD, whilst shrimp injected with commodity inoculate from Vietnam displayed lower grade, albeit characteristic lesions associated with WSSV infection. Animals displaying advanced WSD identified from histopathology were further selected for TEM analysis. Electron microscopy revealed large numbers of rod-shaped virions synonymous with WSSV in epithelial tissues (Fig. 6). Manifestation of WSD in SPF shrimp injected with supermarket and fish market derived commodity products confirmed the viability of WSSV in such products.

Fig. 6. Bioassay of supermarket shrimp in model P. vannamei. Classical pathological signs of WSD were initiated in injected shrimp. WSSV virions were observed in infected epithelial cells, thereby demonstrating off-the-shelf viral viability

Via QPCR, the WSSV-infected shrimp used in the high dose challenge had an equivalent viral loading of 3.65 x 105 copies/ng total DNA. The supermarket-derived commodity shrimp used in the low dose feed challenge had an equivalent viral loading of 5.16 x 102, 4.68 x 101 and 1.04 x 102 copies/ng total DNA in the Honduran, Ecuadorian and Vietnamese shrimp respectively.

Following the feeding of lobsters with high dose products, cumulative mortality of WSSV-fed lobsters in Trial 1 (15oC) reached 5% on day 1 and 10% by day 10 of the trial. In Trial 2 (22oC), cumulative mortality of

EVID4 Evidence Project Final Report (Rev. 06/11) Page 20 of 23 WSSV-fed lobsters reached 40% by Day 3 and 55% by day 6. No further mortalities occurred between day 6 and day 10. Cumulative mortalities in control tanks in Trials 1 and 2 reached 15% by the end of the 10 day trial (Fig. 4). Two moribund or recently dead lobsters in Trial 1 (15oC) and 8 in Trial 2 (22oC) displayed histopathological lesions typical of WSD in other crustacean species, including penaeids. The connective tissues and cuticular epithelium of the limbs appeared to be particularly heavily affected with an apparent loss of tissue mass in these regions and the presence of necrotic cellular debris and remnant WSSV inclusions. Of those lobsters that were fed WSSV-infected material and survived the experimental period, none exhibited the pathologies described above. TEM of lobsters displaying the aforementioned pathologies confirmed the presence of intranuclear inclusions and oval-shaped viral particles, typical of WSSV, within epithelial cell nuclei. Lobster claw tissues used for DNA extraction and nested PCR yielded high quality host genomic DNA as indicated by a characteristic 1800 bp amplification product using the universal SSU rRNA gene primers. All WSSV-challenged lobsters (n=18) held at 15C were positive for WSSV by nested PCR. Eighty % (16/20) of the WSSV-challenged lobsters held at 22C were positive for WSSV by nested PCR. No WSSV-positive results were obtained from the negative control group samples.

Follwing the feeding of lobsters with low-dose supermarket products, cumulative mortality in all treatment tanks varied between groups. Lobsters fed the positive control diet (high-dose WSSV infected P. vannamei) underwent 41% mortality while lobsters in the negative control tank (fed with SPF P. vannamei) underwent 17% mortality. All negative control animals that died during the experiment were undergoing moult at the time of death. Mortality rates in low-dose supermarket-derived commodity fed lobsters were 0%, 20% and 22% for the feed prepared from Honduran, Ecuadorian and Vietnamese shrimp respectively. Histological examination of low-dose fed lobsters did not reveal any of the characteristic signs of WSD as observed in penaeid shrimp or in high-dose fed lobsters. However characteristic signs of WSD were observed once more in the positive control high-dose fed lobsters, particularly in the antennal gland and gill epithelium (as above). TEM of tissues from positive control animals once again revealed the rod-shaped viral particles typical of WSSV within the nucleus of infected cells. Despite the lack of cellular pathology associated with WSD, nested PCR carried out on samples from the low-dose study revealed that almost 100% (16/17) of lobsters fed positive control shrimp were positive for WSSV. In addition, 70% of the lobsters fed supermarket-derived commodity from Honduras displayed positivity in the second round of PCR (sub- patent infection) whilst lobsters fed with supermarket-derived commodity from Ecuador and Vietnam displayed 30% and 45% positivity for WSSV in the second round of PCR, respectively. Sequencing of the nested PCR amplicon from one lobster in each of the treatment tanks followed by analysis using Basic Local Aligned Search Tool (BLAST) confirmed with at least 99% homology that the amplicons were from WSSV (GenBank accession AF332093.1) in all cases tested.

3.4. Implications of viable pathogens in commodity shrimp

As expected from similar surveys in the USA and Australia, a small scale survey of imported penaeid shrimp commodity products from UK supermarkets and fish markets has revealed an apparent widespread prevalence of WSSV-contaminated products. The study demonstrates that frozen commodity shrimp is a route of entry for WSSV into the United Kingdom (and other EU Member States). We have also demonstrated that the WSSV contaminated shrimp tissues contain viable virions that can be passaged to naive susceptible hosts via injection (Litopenaeus vannamei) and feeding (Homarus gammarus). Despite the limitations of the survey design, the underlying within-batch prevalence of the WSSV was relatively high, though in individual components of these batches (e.g. single abdominal sections), the virus was present at a relatively low viral titre, and certainly lower than positive control material generated by passage bioassays to shrimp within our laboratory. This work has also revealed relatively high batch prevalence for WSSV in supermarket-derived commodity, tested using PCR. The apparently higher prevalence in batches of shrimp from supermarkets may represent a higher propensity for supermarket shrimp to be derived from so-called ‟emergency harvest‟ (rapid harvesting of shrimp from culture facilities when it is suspected that a mortality event may be impending). The rapid harvesting of animals essentially averts financial losses but has been suggested to lead to higher viral loading in harvested animals.

Central to any national import risk assessment for commodity products contaminated with pathogens such as WSSV, is an assessment of the likelihood for a pathogen to establish in naive susceptible hosts if the pathogen is released in the receiving location. Feeding trials in which lobsters were fed with low-dose supermarket commodity shrimp demonstrated that passage of WSSV could occur via this natural route and that infection could establish, albeit in latent form, in naive hosts (lobsters) held at temperatures experienced within Europe. Since infection progressed rapidly to disease in lobsters fed with high-dose products (positive control), particularly at higher temperatures, the limiting factor in the rapid appearance of WSD in lobsters is therefore the initial dose; low-level infectious dose establishing latent infection and high- level dose progressing more rapidly to disease. Fundamental studies are now required to assess the potential for latent infections to progress to a disease state and to cause mortality in lobsters retained at European temperatures. Such data are vital for accurate consequence assessment following release and establishment of WSSV infections in wild populations.

EVID4 Evidence Project Final Report (Rev. 06/11) Page 21 of 23

In terms of import risk assessment for the trading of shrimp commodity products, it is known that shrimp imported for human consumption, can also be diverted into alternative uses. The use of raw, frozen shrimp products as angling bait has been identified as a relevant risk for introduction of viral crustacean pathogens in Australia. Furthermore, there is anecdotal evidence (e.g. via online forums) that this practice is also relatively commonplace in the United Kingdom (and likely other parts of Europe). A recent questionnaire sent to a sub-set of the United Kingdom angling fraternity (thought to exceed 4 million individuals) suggested that up to 7% of these may utilise frozen shrimp products as bait (pers. Comm. Dr Birgit Oidtmann). This increased use appears to be directly associated with the current price competitiveness between frozen, imported shrimp commodity and other types of specialist angling bait, and the ease of purchase via supermarkets. Since per os feeding represents a realistic route of entry for pathogens (such as WSSV), further work is now required to investigate the likelihood for pathogen transmission between latently-infected lobster conspecifics and also between infected lobsters and other non-penaeid decapod hosts. Only when such studies are carried out will it be possible to determine the potential for the establishment of WSSV in wild populations of decapods residing in aquatic habitats of temperate regions. Further work is also required to assess the potential for differential risk associated with commodity imported from particular regions, particularly where approaches to within-country biosecurity, or emergency harvesting of infected shrimp ponds, are likely to generate products with high disease status.

EVID4 Evidence Project Final Report (Rev. 06/11) Page 22 of 23

References to published material 9. 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. The following peer-reviewed publications have arisen from work carried out under FC1186. Some of these (publications 1, 2 and 5) relate directly to susceptibility of European crustaceans to the listed pathogens in EC Directive 2006/88. Publications 3 was produced utilising material collected for experimental trials under FC1186. Publication 5 was produced from a literature review of susceptibility of crustaceans to the listed pathogens.

1. Bateman, K.S., Tew, I., French, C., Hicks, R.J., Martin, P., Munro, J., Stentiford, G.D. (2012a). Susceptibility to viral infection and pathogenicity of White Spot Disease (WSD) in non-model crustacean host taxa from temperate regions. Journal of Invertebrate Pathology (in press).

2. Bateman, K.S., Munro, J., Uglow, B., Stentiford, G.D. (2012b). Susceptibility to infection and disease in juvenile European lobster (Homarus gammarus) exposed to high- and low-dose White Spot Syndrome Virus (WSSV) infected shrimp products. Diseases of Aquatic Organisms (in press)

3. Bateman, K.S., Hicks, R.J., Stentiford, G.D. (2011). Disease profiles differ between populations of pre-recruit and recruit edible crabs (Cancer pagurus) from a major commercial fishery. ICES Journal of Marine Science 68, 2044- 2052

4. Stentiford, G.D., Bateman, K.S., Dubuffett, A., Stone, D. (2011). Hepatospora eriocheir (Wang & Chen, 2007) gen. et comb. nov. from European Chinese mitten crabs (Eriocheir sinensis). Journal of Invertebrate Pathology 108, 156-166.

5. Stentiford, G.D., Bonami, J.R., Alday-Sanz, V. (2009). A critical review of susceptibility of crustaceans to Taura syndrome, yellowhead disease and white spot disease and implications of inclusion of these diseases in European legislation. Aquaculture 291, 1-17.

Several other peer-reviewed publications have been produced over the period with support from material and results collected during this project. These include:

6. Feist, S.W., Hine, M., Bateman, K.S., Longshaw, M., Stentiford, G.D. (2009). Paramarteilia canceri n. sp. (Paramyxea) in the European edible crab (Cancer pagurus) with a proposal for the revision of the phylum Paramyxea, Chatton, 1911. Folia Parasitologia 56, 73-85

7. Stentiford, G.D., Bateman, K.S., Small, H.J., Moss, J., Shields, J.D., Reece, K.S., Tuck, I. (2010). Myospora metanephrops (n. gn., n. sp.) from marine lobsters and a proposal for erection of a new Order and Family (Crustaceacida; Myosporidae) in the Class Marinosporidia (Phylum Microsporidia). International Journal for Parasitology 40, 1433-1446.

8. Stentiford, G.D., Neil, D.M. (2011). Diseases of Nephrops and Metanephrops: a review. Journal of Invertebrate Pathology 106, 92-109.

9. Stentiford, G.D. (2011). Diseases of commercially exploited crustaceans: cross-cutting issues for global fisheries and aquaculture. Journal of Invertebrate Pathology 106, 3-5.

10. Stentiford, G.D., Scott, A., Oidtmann, B., Peeler, E. (2010). Crustacean diseases in European legislation: implications for importing and exporting nations. Aquaculture 306, 27-34.

11. Stentiford, G.D., Lightner, D.V. (2011) Cases of White Spot Disease (WSD) in European shrimp farms. Aquaculture 319, 302-306.

12. Small, H.J., Reece, K.S., Shields, J.D., Bateman, K., Stentiford, G.D. (2012). Morphological and molecular characterization of Hematodinium perezi (Dinophyceae: Syndiniales), a dinoflagellate parasite of the harbour crab, Liocarcinus depurator. Journal of Eukaryotic Microbiology 59, 54–66

13. Stentiford G.D., Neil D.M., Peeler E., Shields J.D., Small H.J., Flegel T.W., Vlak J., Jones B., Morado F., Moss, S., Lotz, J., Bartholomay, L., Subasinghe R., Behringer, D.C., Lightner, D.V., Hauton, C., Silva, P. (2012). Disease will limit future food supply from global crustacean fishery and aquaculture sectors. Journal of Invertebrate Pathology (in press).

In addition, we envisage several other publications to arise from the experimental work carried out under FC1186. These publications will be discussed with Defra as they are drafted.

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