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Viral haemorrhagic septicaemia virus (VHSV) and infectious haematopoietic necrosis virus (IHNV) are important viral pathogens posing a serious threat to salmonid fish. Survival of two isolates of IHNV and one of VHSV was assessed at temperatures ranging from 4 to 25°C: (a) after drying on stainless steel, (b) in cell culture medium, (c) in filtered river water, (d) in unfiltered river water, and (e) survival, adsorption and desorption in river sediment and five typical soil types. The viruses survived 1 hr to > 84 days depending on the conditions. Survival was inversely related to temperature and organic and inorganic content. Both viruses remained infectious after being dried on stainless steel for several weeks highlighting the risk of mechanical transmission and persistence in a dry environment. Both adsorbed to the soils from the river water inoculum, with titres between 5.56x104 and 2.58x108 TCID50/ml after 1 hr. Clay soils adsorbed the least virus but had the greatest decrease in the river water inoculum (undetectable in ≤ 1 hr), and there was no desorption. Virus desorbed from the other soils into the surrounding water at different rates dependant on soil type (longest desorption was from chalk loam and sandy soil—detected at 28 days). When desorption was no longer detectable, virus persisted, adsorbed to the soil and remained infectious (the longest adsorption was detected in clay loam for ≥ 49 days, but all the viruses adsorbed to soils were likely to have survived longer than that detected, based on their rate of decay). The long survival of the viruses, particularly at cooler temperatures, highlights the risk of survival in the environment and waterborne spread. The data presented here are highly relevant for assessing risk of pathogen introduction via fomites (stainless steel) and for deciding on best control measures in the context of disease outbreaks.

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.

Infectious hematopoietic necrosis virus (IHNV, Rhabdoviridae), is the causative agent of infectious hematopoietic necrosis (IHN), a disease notifiable to the World Organisation for Animal Health, and various countries and trading areas (including the European Union). IHNV is an economically important pathogen causing clinical disease and mortalities in a wide variety of salmonid species, including the main salmonid species produced in aquaculture, Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss). We reviewed the scientific literature on IHNV on a range of topics, including geographic distribution; host range; conditions required for infection and clinical disease; minimum infectious dose; subclinical infection; shedding of virus by infected fish; transmission via eggs; diagnostic tests; pathogen load and survival of IHNV in host tissues. This information is required for a range of purposes including import risk assessments; parameterisation of disease models; for surveillance planning; and evaluation of the chances of eradication of the pathogen to name just a few. The review focuses on issues that are of relevance for the European context, but many of the data summarised have relevance to IHN globally. Examples for application of the information is presented and data gaps highlighted. Electronic supplementary material The online version of this article (doi:10.1186/s13567-016-0341-1) contains supplementary material, which is available to authorized users.

A qualitative import risk assessment was undertaken to assess the likelihood of introduction and establishment of viral haemorrhagic septicaemia virus (VHSV) genotype 1a in England and Wales (EW), via the processing of imported rainbow trout (Oncorhynchus mykiss) carcasses from continental Europe. The likelihood was estimated for one import from an infected farm. Four main routes by which susceptible populations could be exposed to VHSV via processing waste were considered: (i) run-off from solid waste to watercourses, (ii) contamination of birds or rodents with VHSV by scavenging solid waste, (iii) discharge of liquid waste to mains drainage, and (iv) discharge of liquid waste directly to watercourses. Data on the biophysical characteristics of VHSV, its epidemiology, fish processing practices and waste management were collected. Likelihoods for each step of the four pathways were estimated. Pathway 4 (discharge of liquid waste to a watercourse) was judged as the most likely to result in infection of susceptible individuals. Levels of virus entering the aquatic environment via pathways 1-3 were judged to be many times lower than pathway 4 due mainly to the treatment of solid waste (pathways 1 and 2) and high levels of dilution (pathways 1, 2 and 3). Thirty-four trout farms process fish, of which seven have imported carcasses for processing. Compared with other processing facilities, on-farm processing results in a higher likelihood of VHSV exposure and establishment via all four pathways. Data availability was an issue; the analysis was particularly constrained by a lack of data on the prevalence of VHSV in Europe, volume of trade of carcasses into the UK and processing practices in EW. It was concluded that the threat of VHSV introduction into EW could be reduced by treatment of liquid effluent from processing plants and by sourcing carcasses for on-farm processing only from approved VHSV free areas.

Disposal of fish by-products in the European Community must comply with Regulation (EC) No 1069/2009 which categorizes animal by-products according to risk, and specifies methods of disposal of by-products according to that risk. There is provision under the regulation for composting or ensiling to be used for by-products from aquatic animals. Biosecurity considerations require knowledge of the parameters of time and temperature, or time and pH, required to inactivate any fish pathogens that may be present. To provide those data, we undertook laboratory studies on the inactivation of a number of fish pathogenic viruses and bacteria at 60 °C, pH 4.0 and pH 12.0 as a preliminary to conducting subsequent trials with the most resistant viruses and bacteria in fish tissues. The most resistant bacterium to 60 °C, pH 4.0 as well as pH 12.0 was Lactococcus garvieae. Its concentration was reduced to the level of sensitivity of the test after 24-48 h exposure to 60 °C, but it survived for at least 7 days at pH 4.0 and 14 days at pH 12.0. The most resistant virus to 60 °C was infectious pancreatic necrosis virus, and to pH 12.0 was infectious salmon anaemia virus. The majority of the viruses tested survived exposure to pH 4.0 for up to 28 days. The results suggest that the process of acid ensiling alone is not an effective method for the inactivation of many viral and bacterial pathogens, and fish by-products would need further treatment by a method approved under the regulation following ensiling, whereas alkaline or heat treatment are likely to provide an increased degree of biosecurity for on-farm processing of mortalities.

This study investigated the use of alkaline hydrolysis at ambient temperature for inactivation of selected fish pathogens in fish tissues under conditions approximating those that are likely to be found in the aquaculture industry. Infectious salmon anaemia virus (ISAV) and Lactococcus garvieae have been determined in a previous study to be the most resistant virus and bacteria to pH 12 from a wide range of viruses and bacteria tested. They were spiked at high titres into fish extracts that were then treated with 1 m sodium hydroxide (NaOH). Viable L. garvieae was not detected in the treated fish extract after 1 h, and ISAV was not detected after 24-h exposure. Field mortalities of Atlantic salmon, Salmo salar L., caused by infectious pancreatic necrosis virus were treated by alkaline hydrolysis at ambient temperature. The macerated fish mortalities contained a high titre of virus (3.38 × 10⁸ TCID₅₀ g⁻¹) that was reduced to approximately 2.2 × 10³ TCID₅₀ g⁻¹ after 24-h exposure to NaOH, and virus was not detected after exposure for 48 h. The results suggest that alkaline hydrolysis at ambient temperature has potential as a biosecure treatment method for fish by-products containing fish pathogens.

Market-sized rainbow trout Oncorhynchus mykiss were challenged by waterborne exposure to viral haemorrhagic septicaemia virus (VHSV isolate of genogroup Ia). Fish were sampled at 4 stages of infection (before onset of clinical signs, clinically affected fish, mortalities and survivors) and the viral load determined in (1) internal organs, (2) muscle tissue and (3) brain and gill tissue. Virus levels were determined by virus titration and real-time RT-PCR. VHSV was detected by either method in the majority of fish before onset of clinical signs and in the survivor group as well as in all fish in the clinically affected fish and mortality groups. Mean virus amounts per mg of tissue determined by virus titration (TCID50) or real-time RT-PCR (copy number) were > 10(4) in preclinical fish, > 10(3.8) in clinically affected fish, > 10(3.9) in mortalities and > 10(1.2) in survivors. Virus levels tended to be highest in the internal organs of subclinical and clinically affected fish and in brain and gill tissue of survivors. The results demonstrate that significant levels of VHSV can be found in tissues of rainbow trout that may be marketed for human consumption, which may have relevance for the biosecurity of VHS-free areas.

Movements of commodity fish present a potential risk of transferring pathogens. Within a study to estimate the risk from imported rainbow trout Oncorhynchus mykiss carcases, fry were exposed to tissue homogenates from market size rainbow trout infected experimentally with viral haemorrhagic septicaemia (VHS) by waterborne exposure to VHS virus (VHSV, isolate of genotype Ia). Tissues were collected from fish that showed clinical signs and from recent mortalities. Homogenates of (i) internal organs, (ii) brain/gills and (iii) muscle tissue were prepared and added to tanks holding the fry. Virus transmission occurred from all tissues tested, causing high mortality of the fry. The results underline the potential risk of introduction of VHSV through the trade of fish products.

Estimates of strain effects, heritabilities and genetic correlations for pond survival, resistance to Aeromonas hydrophila and resistance to koi herpesvirus (KHV) were obtained from a diallel cross of 92 full-sib families of common carp produced from four strains (Szarvas 15, Tata, Duna and Amur) and using five females and ten males per strain. Disease resistance was obtained from survival data from challenge-tests using intraperitoneal injection for A. hydrophila and cohabitation for KHV. Two separate challenge-tests were conducted for each disease. The overall survival rates were 44% and 34% for the two tests of A. hydrophila , and 7% and 5% for the two tests of KHV. Pond survival (averaging 78%) was observed over a six months period prior to harvest (at approximately 18 months of age). The three traits were analysed jointly in a multivariate threshold model. For KHV the strain Szarvas 15 had the lowest observed (purebred) survival (0%) followed by Amur (11%), Duna (12%) and Tata (21%), while for A. hydrophila , the lowest (purebred) survival was observed for Duna (28%) followed by Amur (31%), Szarvas 15 (38%) and Tata (48%). Heterosis was not significant for KHV and A. hydrophila resistance, although highly significant for pond survival. The estimated heritability (on the underlying liability scale) was low (0.04 ± 0.03) for A. hydrophila resistance, very high (0.79 ± 0.15) for KHV resistance, and moderate (0.34 ± 0.09) for pond survival. The genetic correlation between the two challenge-tested diseases (KHV and A. hydrophila ) was moderately high (0.61 ± 0.29), although uncertain, while the estimated genetic correlations between pond survival and the two challenge-tested diseases were low and not significantly different from zero (0.01 ± 0.28 and − 0.22 ± 0.21 for A. hydrophila and KHV, respectively). The latter may be expected for KHV, as no outbreaks of the disease had been observed in Hungary. Based on the favourable heritabilities of KHV and pond survival there is good prospect for joint improvement of these two traits in common carps through genetic selection.

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.

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 common carp is an important fish species satisfying ornamental, food and recreational fisheries’ needs worldwide, but in common with other cyprinid fishes, it is particularly renowned for its environmental tolerance. Investigating the mechanistic basis of growth, disease and environmental tolerance is greatly enhanced by access to a comprehensive list of gene sequences and post-genomic technologies. The current status of genomic resources is described for this species including 40 k cDNA clone collections, their associated expressed sequence tags (ESTs) and a developing series of 13 k–26 k cDNA microarrays fabricated from amplicons. The arrays have been directed at questions of response to environmental stress (cold and hypoxia), viral and bacterial disease and ectoparasite infection. Consequently, clones from a wide range of tissues were prepared. The authors discuss how these resources were generated and their application. Evidence is presented supporting that the carp microarray may also be useful as a heterologous set of probes in studies of other fish species.

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.

Several DNA constructs containing the spring viraemia of carp virus (SVCV) glycoprotein (G) gene were investigated for their ability to induce protection against SVCV following injection into myofibres. The constructs were pooled into four groups and co-injected with a plasmid encoding murine granulocyte-macrophage colony-stimulating factor. Group 1 contained one full-length and two truncated G constructs under the control of the cytomegalovirus (CMV) promoter. Group 2 contained full-length constructs with the CMV promoter, the simian virus 40 promoter and a muscle-specific promoter. Group 3 contained constructs in which the G-gene was fused with a second gene in order to improve secretion of the G-protein or to enhance destruction of transfected myocytes by T cells. Group 4 contained constructs with the CMV-Intron A promoter in plasmids with or without CpG motifs. A small-scale trial in goldfish showed that antibody responses in at least half the fish were induced by three injections of plasmids from Groups 1 and 3 whereas T-cell like responses with stimulation indices of above 3 were induced in at least half the fish by Groups 2 and 4. A single-dose of each plasmid mix was then used to protect carp in a large-scale trial. Following challenge with a heterologous strain of SVCV that killed 64% of fish, the strongest protection was observed in carp that received the full length G-gene expressed by two plasmids driven by the CMV-Intron A promoter (Group 4), with a relative percentage survival of 48% (p=0.00008).

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), includ- ing 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. Signifi- cantly, the majority of viruses previously identified as PFRV formed a distinct cluster (Genogroup IV) which shared

Spring viraemia of carp (SVC) is a rhabdovirus infection, which has a significant economic impact in pond cultures of carp in Europe and western Independent States of the former Soviet Union. The causative agent of SVC, spring viraemia of carp virus (SVCV), has been divided into four subgroups, Ia, Ib, Ic and Id, on the basis of glycoprotein (G) protein gene sequences. In this study, a new primer set was designed from a G gene sequence of SVCV to identify the four subtypes of SVCV by reverse transcription polymerase chain reaction (RT-PCR). The specific PCR products of 369 bp were amplified from 15 SVCV isolates of all four subtypes. However, pike fry rhabdovirus (PFRV), which is antigenically related to SVCV, and other viruses antigenically related to SVCV and PFRV were not amplified. The four subtypes of SVCV were specifically amplified by the RT-PCR. Furthermore, the detection limit of the RT-PCR was 7.1 × 10(2) copies/reaction, and it was not influenced by the addition of RNA extracted from fish tissues. The RT-PCR will be applied not only to RNA extracted from viral suspensions, but also from fish tissue. It will contribute to rapid identification of SVCV in fish with clinical signs of SVC.

Flounder, Platichthys flesus L., were exposed to polluted harbour sludge, or to sludge wash-off in mesocosms for three years. Towards the end the study, a pilot experiment was initiated in which flounder antibodies against lymphocystis disease virus were measured in fish held in the mesocosms for 2·5 or 3 years. Although both the sub-sample sizes and number of fish with detectable antibody were small, it was found that between the two sampling periods there was a significant reduction in the number of antibody-positive fish in the mesocosm containing polluted harbour sludge, compared to fish in the mesocosm containing sludge wash-off, or a control mesocosm. The significance of that finding is discussed.

Disposal of fish by-products in the European Community must comply with Regulation (EC) No 1069/2009 which categorizes animal by-products according to risk, and specifies methods of disposal of by-products according to that risk. There is provision under the regulation for composting or ensiling to be used for by-products from aquatic animals. Biosecurity considerations require knowledge of the parameters of time and temperature, or time and pH, required to inactivate any fish pathogens that may be present. To provide those data, we undertook laboratory studies on the inactivation of a number of fish pathogenic viruses and bacteria at 60 °C, pH 4.0 and pH 12.0 as a preliminary to conducting subsequent trials with the most resistant viruses and bacteria in fish tissues. The most resistant bacterium to 60 °C, pH 4.0 as well as pH 12.0 was Lactococcus garvieae. Its concentration was reduced to the level of sensitivity of the test after 24-48 h exposure to 60 °C, but it survived for at least 7 days at pH 4.0 and 14 days at pH 12.0. The most resistant virus to 60 °C was infectious pancreatic necrosis virus, and to pH 12.0 was infectious salmon anaemia virus. The majority of the viruses tested survived exposure to pH 4.0 for up to 28 days. The results suggest that the process of acid ensiling alone is not an effective method for the inactivation of many viral and bacterial pathogens, and fish by-products would need further treatment by a method approved under the regulation following ensiling, whereas alkaline or heat treatment are likely to provide an increased degree of biosecurity for on-farm processing of mortalities.

This study investigated the use of alkaline hydrolysis at ambient temperature for inactivation of selected fish pathogens in fish tissues under conditions approximating those that are likely to be found in the aquaculture industry. Infectious salmon anaemia virus (ISAV) and Lactococcus garvieae have been determined in a previous study to be the most resistant virus and bacteria to pH 12 from a wide range of viruses and bacteria tested. They were spiked at high titres into fish extracts that were then treated with 1 m sodium hydroxide (NaOH). Viable L. garvieae was not detected in the treated fish extract after 1 h, and ISAV was not detected after 24-h exposure. Field mortalities of Atlantic salmon, Salmo salar L., caused by infectious pancreatic necrosis virus were treated by alkaline hydrolysis at ambient temperature. The macerated fish mortalities contained a high titre of virus (3.38 × 10⁸ TCID₅₀ g⁻¹) that was reduced to approximately 2.2 × 10³ TCID₅₀ g⁻¹ after 24-h exposure to NaOH, and virus was not detected after exposure for 48 h. The results suggest that alkaline hydrolysis at ambient temperature has potential as a biosecure treatment method for fish by-products containing fish pathogens.

Koi herpesvirus (KHV) causes a highly virulent disease affecting carp, Cyprinus carpio L., and poses a serious socio-economic threat to the UK carp industry. This study aimed to determine the geographic distribution and prevalence of KHV exposed fish in England and Wales through ELISA antibody testing. Only three of the 82 farms sampled produced positive results, suggesting fish farms provide a relatively safe source of fish. Of the 71 ‘high-risk’ fisheries tested, 26 were positive. All eight geographic areas within England and Wales studied had at least one KHV positive site. Twelve consignments of imported koi carp from seven S.E. Asian countries were tested for KHV antibody. Six consignments from six different countries were positive. Although a high proportion of consignments were positive, the results indicate that lower risk stocks of fish exist that could be sourced by the ornamental carp sector. The study provides evidence that KHV is widespread and prevalent in ‘high-risk’ fisheries. There are, however, prospects for controlling KHV as English and Welsh farms appear to be relatively free of the virus, and in most cases fish are not moved from fisheries to other waters.

Viruses can infect many species of fish and have become most apparent on fish farms. The more virulent viruses can result in haemorrhages, ascites and death. They have been spread between countries by stock fish, ornamental fish, eyed eggs and/or wild fish and so cheap vaccines are still needed to reduce the risk to fish farmers and dealers. However, if infection of fish occurs between vaccination and export then vaccinated excretors can transfer disease between countries. Key concepts: The more serious fish viruses usually result in haemorrhages, ascites and death. New fish viruses continue to be recognized. Fish viruses do not infect man. Fish viruses can spread globally and into new species of fish. Vaccines exist for only a few viruses. The first of all commercial DNA-vaccine was made for infectious haematopoietic necrosis virus of farmed Atlantic salmon in Canada. Vaccines are needed for fry and for the emerging viruses of warm-water fish. Vaccines reduce but do not obviate excretion of virus. Keywords: fish; viruses; vaccines; DNA-vaccine; salmon

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.

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.

The objectives of this study were 1) to describe patterns of use of computed tomography (CT) in laryngeal carcinoma, and 2) to characterize the contribution of CT to the T classification of laryngeal carcinoma.The study population comprised 1195 patients with laryngeal carcinoma diagnosed from 1982 through 1995 chosen randomly from the Ontario provincial cancer registry. A chart review was conducted to obtain data on each case. Patient-related, tumor-related, and health-system-related factors were analyzed to identify factors associated with the use of CT. Descriptions of clinical exams and CT reports were reviewed to see how CT information modified T classification. Actuarial local control and cause specific survival curves were plotted by clinical T classification without and with CT to evaluate stage migration. The percentage of the variance in outcome explained by T classification in a Cox analysis was used to evaluate whether the prognostic accuracy of T classification was improved with the use of information from CT.Patients with glottic (20.1%) and supraglottic (41.7%) carcinoma underwent CT. The use of CT increased over time in glottic and supraglottic carcinoma combined from 17.2% in 1982-5 to 33.9% in 1991-5. Computed tomography was used less often in older patients with a 16% (95% confidence interval, 5-27%) decrease in the odds of having CT with each 10-year age increment. Computed tomography use varied considerably across the cancer center regions in Ontario. Computed tomography altered the T classification in 20.2% of those patients who had CT, with most being "upstages." Stage migration due to CT was demonstrated. Using information from CT in the assignment of T classification for 27.8% of this study population did not make a significant contribution to the ability of T classification to predict outcome over the entire group.There is large variation in the use of CT among different age groups and regions. The ability to compare outcomes by stage across geographic areas is compromised when the use of CT varies.

Squamous cancers of the upper aerodigestive tract (UADT) are related to the use of tobacco and/or alcohol, and in North America they are more common among the poor. They are usually locoregionally confined at diagnosis, and local treatment with surgery and/or radiation therapy is often curative. This study compares the incidence and survival of this group of diseases in Canada and the U.S., two North American neighbors with many cultural similarities but significant differences in their health care and social programs.To describe and compare the case mix, incidence, and outcome of squamous cancers of the UADT in Ontario, Canada, and the U.S., we used the Ontario Cancer Registry (OCR) and the Surveillance, Epidemiology, and End Results (SEER) registries in the U.S. to identify all cases of cancer with International Classification of Disease (ICD) codes 141, 143-9, 160-1, and a subset of 140, which were diagnosed between 1982 and 1994. ICD-O histology codes were placed into clinically relevant groupings, and ICD-9 site codes were grouped into sites as defined by the International Union Against Cancer and the American Joint Committee on Cancer. Age-adjusted incidence rates were calculated for each site. For the SEER registry, race specific incidence rates were also calculated. Observed and expected survival were plotted by site and registry, and from these, relative survival was calculated. Survival was compared during the first 5 years after diagnosis and during the next 5 years among patients who had survived the first 5 years.Of the 16,577 and 42,990 cases identified in the OCR and SEER registries, respectively, squamous cancer was by far the most common histology (94.1% in OCR, 94.6% in SEER) and will form the main subject of this report. The distribution of squamous cancers by site, subsite, age, and gender were remarkably similar in the two populations. Overall, the incidence was about 17% higher in the U.S. than in Ontario, and this difference was seen for all sites except the nasopharynx, which was more common in Ontario. The higher incidence in the U.S. in part reflects the much higher rate for African Americans than for Americans of other ethnic backgrounds. During the first 5 years after diagnosis, when most deaths from UADT cancer occur, there was a significant relative survival difference in favor of the U.S. for cancer of the supraglottis, and in favor of Ontario for cancer of the oral cavity. There was a nonsignificant trend in favor of Ontario for cancer of the nasopharynx. Within the SEER population, for all sites except the nasopharynx, 5-year relative survival was considerably worse for African Americans than for Americans of other ethnic backgrounds. Examination of survival beyond 5 years after diagnosis for patients who had survived the first 5 years revealed that for all sites, the observed survival continued to diverge markedly from the expected survival. The excess mortality ranged from less than 20% for glottic and nasopharyngeal cancers to about 30-40% for oropharyngeal and supraglottic cancers.Despite remarkable similarities in case mix between the two countries, UADT cancers were more frequent in the SEER population of the U.S. than in Ontario, and this was partly attributable to the much higher incidence among African Americans. Significant differences between the registries in 5-year survival were seen for several sites. African Americans with UADT cancers had much worse prognoses than did Americans of other ethnic backgrounds. Patients who survive their UADT cancer remain at a higher-than-expected risk of death even after they have been cured.

To create a classification system for prognosis based on the TNM system for patients with squamous cell carcinoma of the Head and Neck such that patient groupings are homogeneous within and heterogenous between.655 consecutive patients with invasive squamous cell carcinoma of the Head and Neck followed prospectively are split into a training set and a test set. Using the training set, the Cox Proportional Hazards Model and the outcome of dead with disease, we create homogenous prognostic levels based on relative risk. Using the test set, we compare our model to others in the literature.Using the training set we identify 5 prognostic levels and using the test set we demonstrate that our model is superior to others for both within group homogeneity and between group heterogeneity.A simple classification system can be used to group patients for survival and is valid for future study in prognostication.

There is a need for a classification system for prognosis based on the TNM system for patients with squamous cell carcinoma of the head and neck such that patient groupings are homogeneous within and heterogeneous between.Six hundred fifty-five consecutive patients with invasive squamous cell carcinoma of the head and neck followed prospectively are split into a training set and a test set. Using the training set, the Cox Proportional Hazards Model and the outcome of dead with disease, we created homogeneous prognostic levels (PLs) based on RR. Using the test set, we compare our model to others in the literature.Using the training set, we identified five PLs, and using the test set, we demonstrated that our model is superior to others for both within-group homogeneity and between-group heterogeneity.A simple classification system can be used to group patients for survival and is valid for future study in prognostication.

RT-PCR methods have been applied to the detection and sequencing of the glycoprotein gene of viral haemorrhagic septicaemia virus (VHSV), the rhabdovirus which causes viral haemorrhagic septicaemia (VHS) in farmed salmonid fish. Phylogenetic analysis of a 360 nt region of the glycoprotein gene from a range of marine and fresh water VHSV isolates identified three genogroups, I-III. Significantly, two virus isolates recovered from ulcerated North Sea cod caught off the Shetland Islands, and an isolate recovered from diseased turbot farmed on the island of Gigha, Scotland were assigned to the same genogroup. Moreover, a virus isolated from diseased turbot farmed on the Baltic Sea coast shared 99.4% nucleotide sequence similarity with a virus associated with a VHS outbreak in rainbow trout. This is the first time that a genetic link between a VHS outbreak and natural VHSV infections of marine fish species has been demonstrated.

To examine the relationship between N stage and survival in head and neck cancer to see if N stage has prognostic value.Database analysis using the Cox Proportional Hazards Model.Five hundred and twenty-three consecutive patients with squamous cell carcinoma of the head and neck prospectively entered into a clinical database are analyzed for the outcome of dead with disease.There is no difference in survival between some increments of N stage, specifically N1 and N2, controlling for T stage and site.A modified N stage system consisting of N0, N(limited), and N(extended) appears to have a better prognostic value.

A clinical database documents caseload, assesses treatments, compares treatments, follows patients, and can be used for effective teaching. The database and software used at Kingston Regional Cancer Clinic are described, and a few pertinent findings are presented.

Exploratory studies into the immunocompetence of fish held on contaminated and uncontaminated bottom sediments have been conducted at the Fish Diseases Laboratory, Weymouth. The approach has been to expose dab, Limanda limanda L. (Pleuronectidae) long-term (> 3 months) on highly contaminated harbour sludge and less-contaminated sediment, and to compare the immune response and uptakes of contaminants between the two groups. Following exposure to the sediments, mortalities and clinical signs of disease were recorded. The dab were then challenged individually by intraperitoneal injection with 0–1 ml of a 1% suspension of inactivated Vibrio anguillarum . The fish were examined for clinical signs of disease. Blood and samples of organs, including brain, were taken for histological examination. The remainder of these fish (and all fish dying during the experiments) were frozen for contaminant uptake analysis. Clinical and histopathological changes were observed in both groups of fish with skin ulcerations and gill hyperplasia, showing a slightly higher prevalence in the fish exposed to contaminated harbour sludge. Quantification of the immune response was by agglutination assay and an enzyme-linked immunosorbent assay (ELISA). Fish maintained on the contaminated harbour sludge had a lower mean antibody titre than those on the reference sediment and this difference in mean titres was statistically significant. However, because the fish on the contaminated sediment were still able to produce a marked response to the antigen, in biological terms there may have been no difference in the susceptibility to disease between the two groups. Further work will be needed to clarify this point.

The results of treatment are reviewed in 18 cases of childhood nasopharyngeal carcinoma. Since 1976 adjuvant chemotherapy with cyclophosphamide, methotrexate, and adriamycin has been used in Manchester and Leeds. The outcome is compared in 9 patients treated with radiotherapy alone and 9 patients treated later with both radiotherapy and adjuvant chemotherapy. Relapse-free survival rates were 0 % for those treated with radiotherapy alone and 78% at 13–80 months after diagnosis for those treated with both radiotherapy and chemotherapy. It is concluded that adjuvant chemotherapy improves relapse-free survival in childhood nasopharyngeal carcinoma.

Summary The optimum conditions for staphylococcal co-agglutination (CoA), latex agglutination (LA) and the fluorescent antibody test (FAT) for the identification of Renibacterium salmoninarum isolated in culture were determined. Their sensitivities were then compared with that of the enzyme-linked immunosorbent assay (ELISA). The ELISA was the most sensitive, followed by CoA, with the FAT and LA jointly the least sensitive. However, all four techniques were sufficiently sensitive for routine identification of R. salmoninarum. The agglutination techniques (CoA and LA) were preferred because of their speed, ease of use and low cost.

Summary An enzyme-linked immunosorbent assay (ELISA) was developed for the detection and identification of Renibacterium salmoninarum. The immune γ-globulin used in the assay was absorbed with two species of cross-reacting bacteria to make a specific test system. R. salmoninarum could be detected in clinically-diseased fish within 30 minutes of preparing a kidney sample, and thus because of its ease of use, the ELISA could be employed as a rapid field test for bacterial kidney disease (BKD), although isolation of R. salmoninarum was more sensitive than the ELISA for detecting individual carrier fish.

The ELISA technique was adapted for the rapid identification of infectious haematopoietic necrosis virus and two eel rhabdoviruses in cell cultures or directly from tissues of infected fry. Results were obtained in under 2 h. The assays were specific for the viruses when infected cell cultures were tested, but there were low-level cross-reactions with certain other rhabdoviruses when the test was applied to extracts of infected fry. Attempts to produce assays for the detection of viral haemorrhagic septicaemia virus and spring viraemia of carp virus were unsuccessful, apparently because the titres of the antisera were too low.

Formalin and β-propiolactone (BPL) were compared for efficacy in inactivating infectious pancreatic necrosis virus (IPNV) for vaccine use. Incubation with a 1:200 dilution of formalin at 20°C for four days or longer, or a 1:200 dilution of BPL at 4°C for six days or longer, completely inactivated the virus infectivity. However, whereas treatment with formalin caused only a slight reduction in anti-genicity as titrated by in vitro tests, treatment with BPL destroyed over 50% of the antigenicity of the virus. Virus inactivated with formalin also proved highly effective at inducing high titres of neutralizing antibody in trout.

Summary Antisera raised in rabbits against viral haemorrhagic septicaemia virus (VHSV) and infectious haematopoietic necrosis virus (IHNV) contained high levels of binding antibody which made possible the development of rapid immunoassays. These were used to detect VHS and IHN viral antigen in cell culture and in extracts of infected fry. Virus could be detected in cell culture before any cytopathic effect was noticed. The ELISA for VHSV was most sensitive and detected antigens in cell culture after only 12 h. Both assays were specific for their respective viruses and results were obtained in under 2 h. Zusammenfassung Der Schnellnachweis von VHS-und IHN-Viren mittels des ELISA (enzyme-linked immunosorbent assay) Von Kaninchen gewonnene Antiseren gegen das Virus der viralen hamorrhagischen Septikamie (VHSV) und das Virus der infektiosen hamatopoetischen Nekrose (IHNV) enthielten hohe Anteile bindender Antikorper, die die Entwicklung schneller Immunreaktionen ermoglichten. Sie wurden zum Nachweis von VHS-und IHN-Virusantigen in Zeukulturen und Extrakten aus infizierten Fischen verwendet. Viren konnten in Zellkulturen nachgewiesen werden, noch ehe irgendwelche zytopathischen Effekte zu beobachten waren. Der ELISA auf VHSV war am empfindlichsten, das Antigen konnte in Zellkulturen schon nach 12 Stunden nachgewiesen werden. Die Ergebnisse des virusspezifischen Testverfahrens lagen bereits nach weniger als zwei Stunden vor. Resume Le decelement rapide des virus de SH V et de NHI par le test immunosorbant lie aux enzymes (ELISA) Des antisera obtenus sur le lapin afin de lutter contre la septicemic hemorragique virale (SHV) et le virus de la necrose hematopoietique infectieuse se sont averes contenir de hautes teneurs en anticorps liants qui ont permis d'elaborer des tests immunologiques rapides. Ces tests ont servi a deceler les antigenes viraux de la SHV et de la NHI dans des cultures de cellules ainsi que dans des extraits de nourrains. Le virus a pu etre decele dans la culture de cellules avant qu'un quelconque phenomene cytopathique ne soit observe. L'ELISA (test immunosorbant lie aux enzymes) de la SHV s'est averee la plus sensible a deceler des antigenes dans la culture de cellules dans un delai de 12 heures seulement. Les deux tests sont specifiques a leurs virus respectifs et des resultats ont ete obtenus en moins de 2 heures.