A seafood risk tool for assessing and mitigating chemical and pathogen hazards in the aquaculture supply chain.

Intricate links between aquatic animals and their environment expose them to chemical and pathogenic hazards, which can disrupt seafood supply. Here we outline a risk schema for assessing potential impacts of chemical and microbial hazards on discrete subsectors of aquaculture-and control measures that may protect supply. As national governments develop strategies to achieve volumetric expansion in seafood production from aquaculture to meet increasing demand, we propose an urgent need for simultaneous focus on controlling those hazards that limit its production, harvesting, processing, trade and safe consumption. Policies aligning national and international water quality control measures for minimizing interaction with, and impact of, hazards on seafood supply will be critical as consumers increasingly rely on the aquaculture sector to supply safe, nutritious and healthy diets.

Genetic diversity and associated pathology of rhabdovirus infections in farmed and wild perch Perca fluviatilis in Ireland.

Rhabdovirus infections are an emerging problem for both wild and farmed freshwater fish in Northern Europe. In October 2005, a clinical outbreak with an approximate mortality rate of 40% occurred in a single batch of juvenile perch on a farm in the Republic of Ireland. Clinical signs developed slowly and were consistent with a perch rhabdovirus infection: signs included haemorrhages at the base of the fins and apparent impairment of the central nervous system (manifested as loss of equilibrium and erratic swimming behaviour). Studies suggest that the infected fish originated from a hatchery within the country which relied on wild fish broodstock to supplement the production of perch juveniles. A related rhabdovirus was subsequently isolated from this hatchery. Virus isolation studies have shown that rhabdoviruses were often isolated from wild fish in the vicinity of the hatchery between 1993 and 2005. All isolates were analysed using a generic primer set specific for the L gene of fish vesiculotype viruses. Phylogenetic analysis revealed that all isolates recovered from perch clustered together with the European lake trout rhabdovirus (903/87) of the genus Perhabdovirus. In addition to this, anguillid rhabdovirus was isolated from eel, and the partial L-gene sequence of a previously reported isolate from tench clustered with the pike fry rhabdoviruses, in the genus Sprivivirus.

Areospora rohanae n.gen. n.sp. (Microsporidia; Areosporiidae n. fam.) elicits multi-nucleate giant-cell formation in southern king crab (Lithodes santolla).

This paper utilises histological, ultrastructure and molecular phylogenetic data to describe a novel genus and species (Areospora rohanae n.gen., n.sp.) within the phylum Microsporidia. Phylogenetic and morphological distinction from other known lineages within the phylum also provide strong support for erection of a new family (Areosporiidae n. fam) to contain the parasite. Recognised via lesions observed by workers in king crab processing facilities in southern Chile, the parasite elicits giant cell formation in infected crabs. Merogony within haemocytes and fixed phagocytes proceeds apparent fusion of infected cells to produce multinucleate syncitia in which further development of the parasite occurs. Subsequent recruitment of adjacent cells within the haemal spaces of the hepatopancreas, the podocytes of the gill, and particularly in the subcuticular connective tissues, characterises the pathogenesis of A. rohanae. In late stages of infection, significant remodelling of the subcuticular tissues corresponds to the clinical lesions observed within processing plants. Sporogony of A. rohanae also occurs within the syncitial cytoplasm and culminates in production of bizarre spores, ornamented with distinctive tubular bristles. Spores occur in sets of 8 within a sporophorous vesicle. The description of A. rohanae offers considerable insight into the pathogenesis of giant-cell forming Microsporidia, signifies a new lineage of giant-cell forming Microsporidia in marine hosts, and may reflect emergence of a commercially-significant pathogen in the southern ocean Lithodes santolla fishery.

Spring viraemia of carp (SVC) in the UK: the road to freedom.

Spring viraemia of carp (SVC) is a disease of international importance that predominantly affects cyprinid fish and can cause significant mortality. In the United Kingdom (UK), SVC was first detected in 1977 with further cases occurring in fisheries, farms, wholesale and retail establishments throughout England and Wales (but not Scotland, where few cyprinid populations exist, nor Northern Ireland where SVC has never been detected) over the subsequent 30 years. Following a control and eradication programme for the disease initiated in 2005, the UK was recognised free of the disease in 2010. This study compiles historic records of SVC cases in England and Wales with a view to understanding its routes of introduction and spread, and assessing the effectiveness of the control and eradication programme in order to improve contingency plans to prevent and control future disease incursions in the cyprinid fish sectors. Between 1977 and 2010 the presence of SVC was confirmed on 108 occasions, with 65 of the cases occurring in sport fisheries and the majority of the remainder occurring in the ornamental fish sector. The study found that throughout the history of SVC in the UK, though cases were widely distributed, their occurrence was sporadic and the virus did not become endemic. All evidence indicates that SVC was not able to persist under UK environmental conditions, suggesting that the majority of cases were a result of new introductions to the UK as opposed to within-country spread. The control and eradication programme adopted in 2005 was highly effective and two years after its implementation cases of SVC ceased. Given the non-persistent nature of the pathogen the most important aspect of the control programme focused on preventing re-introduction of the virus to the UK. Despite the effectiveness of these controls against SVC, this approach is likely to be less effective against more persistent pathogens such as koi herpesvirus, which are likely to require more stringent measures to prevent within-country spread.

Plastic parasites: extreme dimorphism creates a taxonomic conundrum in the phylum Microsporidia.

In this paper, we combine field observations of highly statistically significant co-occurrence with histopathological, ultrastructural and molecular phylogenetic analyses, to provide evidence for extreme morphological plasticity in a microsporidium parasite infecting the musculature of marine crabs. The parasite appears to alternate between lineages that culminate in production of either bizarre needle-like spores in the peripheral sarcoplasm of heart and skeletal muscle fibres (reminiscent of Nadelspora canceri infecting Cancer magister) or alternatively, Ameson-like spores with pronounced surface projections, in the skeletal muscles (as for Ameson pulvis, previously described infecting Carcinus maenas). Both lineages occur in direct contact with the cytoplasm of host muscle cells and can exist simultaneously within the same cell. Pathological data appears to reveal a remarkable shift in morphology during pathogenic remodelling of host tissues. Sequence analysis of multiple clones derived from amplification of the ssrRNA gene from infected regions of the heart and skeletal muscles appear to confirm the genetic identity of the two lineages. Furthermore, derived ssrRNA gene sequences are more similar (>99%) to N. canceri than to the coparasite Ameson michaelis infecting Callinectes sapidus (93%). Although molecular phylogenetic data support transfer of A. pulvis into the genus Nadelspora, the expansion in the generic description required to include such widely divergent characteristics is so significant as to be unfeasible within the current taxonomic framework of the phylum Microsporidia. At present, it is preferable to propose that the parasite infecting C. maenas forms a clade with other morphologically diverse but phylogenetically and ecologically similar muscle-infecting microsporidians from marine crustacean hosts. Given the strong evidence for significant plasticity in morphology amongst members of the phylum Microsporidia, novel approaches to phylogeny, based predominantly upon the informed use of molecular sequence data, are now deemed a necessity.

Hepatospora eriocheir (Wang and Chen, 2007) gen. et comb. nov. infecting invasive Chinese mitten crabs (Eriocheir sinensis) in Europe.

We describe a microsporidian parasite infecting non-native Chinese mitten crabs (Eriochier sinensis) from Europe. Electron microscopy revealed merogonic and sporogonic life stages bound within a plasmalemma. The crab parasite develops polar tube precursors at the sporont stage but does not complete formation of the intact spore extrusion apparatus at the stage of the sporogonial plasmodium like Enterocytozoon bienuesi and other representatives of the Enterocytozoonidae. Its presence within an aquatic crustacean host, and a distinct molecular phylogeny based on partial small subunit ribosomal RNA (SSU rRNA) gene sequences also place it relatively close, though distinct to, existing genera within the Enterocytozoonidae. Consideration of morphological and phylogenetic characteristics of other hepatopancreas-infecting microsporidia from crustaceans suggests that certain ones (e.g. Enterospora canceri) are retained within the clade corresponding to the existing family Enterocytozoonidae, while others, including the parasite described here, may eventually be grouped in a sister taxon potentially of family rank. Based upon morphological and host similarity, it is likely that the parasite described here is the same as Endoreticulatus eriocheir (Wang and Chen, 2007), previously described from Chinese mitten crabs in Asia. However, using a combined taxonomic approach based upon morphological and phylogenetic data, we propose the formation of a new genus (Hepatospora) to replace the previous generic classification of the Asian parasite as Endoreticulatus. The microsporidian from the hepatopancreas of E. sinensis is named Hepatospora eriocheir (Wang and Chen, 2007) gen. et comb. nov. It is assumed that the parasite was introduced during initial invasions of this crab to Europe during the early 20th Century.

Multi-centre testing and validation of current protocols for the identification of Gyrodactylus salaris (Monogenea).

Despite routine screening requirements for the notifiable fish pathogen Gyrodactylus salaris, no standard operating procedure exists for its rapid identification and discrimination from other species of Gyrodactylus. This study assessed screening and identification efficiencies under real-world conditions for the most commonly employed identification methodologies: visual, morphometric and molecular analyses. Obtained data were used to design a best-practice processing and decision-making protocol allowing rapid specimen throughput and maximal classification accuracy. True specimen identities were established using a consensus from all three identification methods, coupled with the use of host and location information. The most experienced salmonid gyrodactylid expert correctly identified 95.1% of G. salaris specimens. Statistical methods of classification identified 66.7% of the G. salaris, demonstrating the need for much wider training. Molecular techniques (internal transcribed spacer region-restriction fragment length polymorphism (ITS-RFLP)/cytochrome c oxidase I (COI) sequencing) conducted in the diagnostic laboratory most experienced in the analysis of gyrodactylid material, identified 100% of the true G. salaris specimens. Taking into account causes of potential specimen loss, the probabilities of a specimen being accurately identified were 95%, 87% and 92% for visual, morphometric and molecular techniques, respectively, and the probabilities of correctly identifying a specimen of G. salaris by each method were 81%, 58% and 92%. Inter-analyst agreement for 189 gyrodactylids assessed by all three methods using Fleiss' Kappa suggested substantial agreement in identification between the methods. During routine surveillance periods when low numbers of specimens are analysed, we recommend that specimens be analysed using the ITS-RFLP approach followed by sequencing of specimens with a "G. salaris-like" (i.e. G. salaris, Gyrodactylus thymalli) banding pattern. During periods of suspected outbreaks, where a high volume of specimens is expected, we recommended that specimens be identified using visual identification, as the fastest processing method, to select "G. salaris-like" specimens, which are subsequently identified by molecular-based techniques.

Proposal for a fourth aquabirnavirus serogroup.

Four putative aquabirnaviruses, based on morphology, nucleic acid type and partial RNA-dependent RNA polymerase gene (VP1) sequence, isolated from three tropical freshwater fish species were not neutralised by antisera against type members of the Aquabirnavirus genus serogroups A, B or C. Antisera produced against two of the isolates neutralised the homologous and heterologous isolates, but not any type member of Aquabirnavirus serogroups A, B or C. The serological comparisons suggest that the four isolates should be regarded as members of a fourth Aquabirnavirus serogroup, D.

The first report of viral haemorrhagic septicaemia in farmed rainbow trout, Oncorhynchus mykiss (Walbaum), in the United Kingdom.

Viral haemorrhagic septicaemia (VHS) was diagnosed in rainbow trout in the UK in May 2006. VHS virus (VHSV) was isolated from fingerlings showing typical histopathological lesions at a single rainbow trout farm site experiencing high mortality. The virus was confirmed as VHSV by serological and molecular biological tests. Phylogenetic analysis based on the complete glycoprotein gene sequence revealed that the isolate was closely related (99% nucleotide identity) to several Danish isolates from 1991 to 2000 and was assigned to VHSV genogroup Ia. The pathogenicity of the isolate was determined in infection experiments using rainbow trout fry. Following waterborne challenge, cumulative mortalities reached 96.67-100% by 12 days post-infection. This represents the first isolation of a pathogenic freshwater VHSV in the UK.

Assessing the environmental risk from hot particles in the vicinity of Dounreay--a case for inaction?

This study assesses the impact on species other than humans associated with radioactive particles present in the marine environment close to the UKAEA Dounreay site, through a review of marine survey data, to establish the distribution of species and the likelihood of encountering a particle, and considering retention, dissolution or absorption of the particle. Assumptions are made regarding particle density, distribution, size and bio-availability of the radioactive materials. From this, impacts are assessed against the likelihood of mortality or other significant harm to individuals and interpreted in terms of local populations. Results obtained indicate that no significant impact, at the population level, is likely to be observed. This does not preclude that some individuals will be affected. It does, however, suggest that any decision to remediate, if based predominantly on environmental considerations, should be cognisant of the damage caused by remediation itself and subsequent exploitation of the environment by humans.

Phylogenetic analysis of spring virema of carp virus reveals distinct subgroups with common origins for recent isolates in North America and the UK.

Genetic relationships between 35 spring viremia of carp virus (SVCV) genogroup Ia isolates were determined based on the nucleotide sequences of the phosphoprotein (P) gene and glycoprotein (G) genes. Phylogenetic analysis based on P gene sequences revealed 2 distinct subgroups within SVCV genogroup Ia, designated SVCV Iai and Iaii, and suggests at least 2 independent introductions of the virus into the USA in 2002. Combined P- and G-sequence data support the emergence of SVCV in Illinois, USA, and in Lake Ontario, Canada, from the initial outbreak in Wisconsin, USA, and demonstrate a close genetic link to viruses isolated during routine import checks on fish brought into the UK from Asia. The data also showed a genetic link between SVCV isolations made in Missouri and Washington, USA, in 2004 and the earlier isolation made in North Carolina, USA, in 2002. However, based on the close relationship to a 2004 UK isolate, the data suggest than the Washington isolate represents a third introduction into the US from a common source, rather than a reemergence from the 2002 isolate. There was strong phylogenetic support for an Asian origin for 9 of 16 UK viruses isolated either from imported fish, or shown to have been in direct contact with fish imported from Asia. In one case, there was 100% nucleotide identity in the G-gene with a virus isolated in China.

Genotyping spring viraemia of carp virus and other piscine vesiculo-like viruses using reverse hybridisation.

A simple nylon membrane-based DNA macroarray was developed to genotype spring viraemia of carp virus (SVCV) and related viruses. Twenty-six viruses were genotyped using the array, and the results were confirmed by phylogenetic analysis of a 426 bp partial glycoprotein gene sequence. The array was not only capable of discriminating between the 4 main genogroups of cyprinid vesiculo-type viruses described previously, but also accurately sub-type the SVC viruses assigned to Genogroup I. The assay offers a practical solution for diagnostic laboratories that currently lack a sequencing capability to confirm the nature of PCR products generated in suspected SVCV cases.

Experimental Rhodococcus equi and equine infectious anemia virus DNA vaccination in adult and neonatal horses: effect of IL-12, dose, and route.

Improving the ability of DNA-based vaccines to induce potent Type1/Th1 responses against intracellular pathogens in large outbred species is essential. Rhodoccocus equi and equine infectious anemia virus (EIAV) are two naturally occurring equine pathogens that also serve as important large animal models of neonatal immunity and lentiviral immune control. Neonates present a unique challenge for immunization due to their diminished immunologic capabilities and apparent Th2 bias. In an effort to augment R. equi- and EIAV-specific Th1 responses induced by DNA vaccination, we hypothesized that a dual promoter plasmid encoding recombinant equine IL-12 (rEqIL-12) would function as a molecular adjuvant. In adult horses, DNA vaccines induced R. equi- and EIAV-specific antibody and lymphoproliferative responses, and EIAV-specific CTL and tetramer-positive CD8+ T lymphocytes. These responses were not enhanced by the rEqIL-12 plasmid. In neonatal foals, DNA immunization induced EIAV-specific antibody and lymphoproliferative responses, but not CTL. The R. equi vapA vaccine was poorly immunogenic in foals even when co-administered with the IL-12 plasmid. It was concluded that DNA immunization was capable of inducing Th1 responses in horses; dose and route were significant variables, but rEqIL-12 was not an effective molecular adjuvant. Additional work is needed to optimize DNA vaccine-induced Th1 responses in horses, especially in neonates.

Altered prostaglandin E1 dosage during extracorporeal membrane oxygenation in a newborn with ductal-dependent congenital heart disease.

We describe a neonate with ductal-dependent congenital heart disease on extracorporeal membrane oxygenation (ECMO) for persistent pulmonary hypertension, who required markedly high doses of prostaglandin E1 (PGE1) to maintain patency of the ductus arteriosus: The effects of ECMO on the pharmacokinetics of PGE1 are discussed.

Four years of monitoring for viral haemorrhagic septicaemia virus in marine waters around the United Kingdom.

Between 1995 and 1998, marine fish from around the coast of the UK were collected and samples analysed for viral haemorrhagic septicaemia virus (VHSV) using cell culture isolation methods. In 1997 and 1998 the samples were also analysed for VHSV by reverse transcription PCR (RT-PCR). A total of 1867 fish of 11 species were tested, but VHSV was isolated on only 1 occasion, from herring Clupea harengus, in 1996. However, despite VHSV not being isolated in 1997 and 1998, in both years samples of herring from the west and south coasts of England produced positive signals in the RT-PCR, and in 1997 cod from the east coast of England also produced positive signals in the RT-PCR. These results are believed to be true indications of the presence of VHSV nucleic acid in the fish. In 1997, birnaviruses from Serogroup B1 were isolated from herring (a previously unrecorded host for the virus) and cod Gadus morhua, and a birnavirus from Serogroup A2 was also isolated from cod. In 1998, an aquareovirus was isolated from haddock Melanogrammus aeglefinus, a previously unrecorded host for the virus.

Nucleotide sequence analysis of the glycoprotein gene of putative spring viraemia of carp virus and pike fry rhabdovirus isolates reveals four genogroups.

RT-PCR methods have been applied to the detection and sequencing of the glycoprotein gene of putative spring viraemia of carp viruses (SVCV) and pike fry rhabdoviruses (PFRV), including isolates from tench, grass carp, roach, bream and false harlequin, sheatfish and orfe. Phylogenetic analysis of a 550 nucleotide (nt) region of the glycoprotein gene identified 4 groups, I to IV. Significantly, the majority of viruses previously identified as PFRV formed a distinct cluster (Genogroup IV) which shared <80% nucleotide identity with the PFRV reference strain (Genogroup III). The similarity between another PFRV-like virus isolated from grass carp and representatives of Genogroups III and IV was also <80%, indicating that this virus belonged to a third group (Genogroup II). All of the putative SVC viruses were assigned to a 4th group (Genogroup I), sharing <61% nucleotide identity with viruses in Genogroups II to IV.

Lethal and sublethal effects of a neem-based insecticide on balsam fir sawfly (Hymenoptera: Diprionidae).

Lethal and sublethal effects of Neemix 4.5 EC, a commercial neem preparation, on balsam fir sawfly, Neodiprion abietis (Harris), were determined in the laboratory. Larval mortality of N. abietis increased in a concentration-dependent manner, and lethal time decreased with increasing Neemix 4.5 EC concentration. Fifty percent of the larvae died after 4.6 d at a concentration of 90 ppm azadirachtin (AZA) and 12.3 d at a concentration of 0.08 ppm. Neemix 4.5 EC showed some deterrent effects to feeding site selection on N. abietis larvae at high concentrations, but not at low concentrations. Strong reduction of food intake by N. abietis larvae after exposure to Neemix 4.5 EC was demonstrated by significant reduction of frass production. Larvae fed on Neemix 4.5 EC-treated foliage at 90 ppm AZA produced only 16% as much frass as that produced by larvae fed on control foliage (0 ppm). Neemix 4.5 EC at a concentration of 0.08 ppm AZA retarded larval and pupal development. Sublethal doses significantly reduced pupal weight and adult emergence, although the sex ratio of N. abietis adults was not affected. Results indicate that sublethal effects of Neemix 4.5 EC on N. abietis may contribute greatly to the overall field efficacy.

Emerging vesiculo-type virus infections of freshwater fishes in Europe.

Rhabdoviruses were isolated from perch Perca fluviatilis and largemouth bass Micropterus salmoides exhibiting clinical signs of disease. Preliminary studies indicated that these viruses could be neutralised by antisera to perch rhabdovirus (Dorson et al. 1984) and may be similar to those previously isolated from grayling Thymallus thymallus and pike-perch Stizostedion stizostedion. The relationship between these viruses and the previously characterised fish rhabdoviruses, pike fry rhabdovirus (PFRV), spring viraemia of carp virus (SVCV) and lake trout rhabdovirus, was investigated. Viruses were propagated in bluegill fry (BF-2) cells and were characterised using electron microscopy, serum neutralisation tests, immunofluorescence tests, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and nucleotide sequence analysis. The bullet-shaped viral particles appeared to be compact, with spikes visible at the surface, a morphology similar to that of the vesiculovirus group of rhabdoviruses. Serum neutralisation tests showed that the viruses were antigenically closely related to the previously characterised perch rhabdovirus, but were not significantly neutralised by antisera to PFRV, SVCV or viral haemorrhagic septicaemia virus (VHSV). In immunofluorescence tests with perch rhabdovirus antisera, strong specific fluorescence was observed in cell cultures infected with the new rhabdovirus isolates, but no fluorescence was observed with antisera to PFRV, SVCV or VHSV. SDS-PAGE analysis revealed a polypeptide profile typical of vesiculoviruses, but the novel virus isolates had different relative mobilities of their P and M proteins compared to PFRV and SVCV. Nucleotide sequence analysis was carried out using reverse transcriptase-polymerase chain reaction (RT-PCR) and DNA sequencing of a 439 base-pair region of the viral L gene. The novel rhabdovirus isolates had <76% nucleotide sequence identity to PFRV, SVCV and lake trout rhabdovirus and >95% identity to perch rhabdovirus. Phylogenetic analysis using both maximum parsimony and neighbour-joining methods assigned the perch rhaboviruses to a separate group to that of PFRV, SVCV and lake trout rhabdovirus. These data are the initial characterisation of a group of emerging fish vesiculo-type viruses that are biochemically and genetically distinct from the PFRV, SVCV and lake trout rhabdoviruses.

Isolation of a rhabdovirus during outbreaks of disease in cyprinid fish species at fishery sites in England.

A virus was isolated during disease outbreaks in bream Abramis brama, tench Tinca tinca, roach Rutilis rutilis and crucian carp Carassius carassius populations at 6 fishery sites in England in 1999. Mortalities at the sites were primarily among recently introduced fish and the predominant fish species affected was bream. The bream stocked at 5 of the 6 English fishery sites were found to have originated from the River Bann, Northern Ireland. Most fish presented few consistent external signs of disease but some exhibited clinical signs similar to those of spring viraemia of carp (SVC), with extensive skin haemorrhages, ulceration on the flanks and internal signs including ascites and petechial haemorrhages. The most prominent histopathological changes were hepatocellular necrosis, interstitial nephritis and splenitis. The virus induced a cytopathic effect in tissue cultures (Epithelioma papulosum cyprini [EPC] cells) at 20 degrees C and produced moderate signals in an enzyme immunoassay (EIA) for the detection of SVC virus. The virus showed a close serological relationship to pike fry rhabdovirus in both EIA and serum neutralisation assays and to a rhabdovirus isolated during a disease outbreak in a bream population in the River Bann in 1998. A high degree of sequence similarity (> or = 99.5% nucleotide identity) was observed between the English isolates and those from the River Bann. Experimental infection of juvenile bream, tench and carp with EPC cell-grown rhabdovirus by bath and intraperitoneal injection resulted in a 40% mortality of bream in the injection group only. The virus was re-isolated from pooled kidney, liver and spleen tissue samples from moribund bream. The field observations together with the experimental results indicate that this rhabdovirus is of low virulence but may have the potential to cause significant mortality in fishes under stress.

Rescue of viral haemorrhagic septicaemia virus minigenomes by helper virus.

A mammalian expression vector containing the bacterial chloramphenicol acetyltransferase (CAT) gene was used to demonstrate that CAT could be successfully used as a reporter system in fish cells growing at low temperatures. We then constructed a viral haemorrhagic septicaemia virus (VHSV) minigenome by cloning the CAT reporter gene between the viral leader and trailer sequences. This construct was used in transfection experiments with helper VHSV to demonstrate that the minigenome can be encapsidated and transcribed by helper virus proteins. In addition, passaging of viruses collected from cells expressing the minigenome showed that the minigenome was being packaged and replicated in the presence of helper virus. These experiments provide the initiating steps for a reverse genetics system for VHSV.