Journal of Fish DiseasesVolume 35, Issue 10 p. 789-792 Short Communication The stability of infectious salmon anaemia virus infectivity at −80 °C in tissue homogenate and dry-stored tissue from clinically diseased Atlantic salmon, salmo salar L. D A Smail, Corresponding Author D A Smail Marine Laboratory, Marine Scotland, Aberdeen, UKCorrespondence D A Smail, Marine Scotland, Marine Laboratory, PO Box 101, 375 Victoria Road, Aberdeen, AB11 9DB (e-mail: david.smail@scotland.gsi.gov.uk)Search for more papers by this authorR Grant, R Grant Silberline Ltd, Fife, UKSearch for more papers by this author D A Smail, Corresponding Author D A Smail Marine Laboratory, Marine Scotland, Aberdeen, UKCorrespondence D A Smail, Marine Scotland, Marine Laboratory, PO Box 101, 375 Victoria Road, Aberdeen, AB11 9DB (e-mail: david.smail@scotland.gsi.gov.uk)Search for more papers by this authorR Grant, R Grant Silberline Ltd, Fife, UKSearch for more papers by this author First published: 17 July 2012 https://doi.org/10.1111/j.1365-2761.2012.01402.xCitations: 1 This article is published with the permission of the Controller of HMSO and the Queen's Printer for Scotland. Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume35, Issue10October 2012Pages 789-792 RelatedInformation

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 chapter contains sections titled: Health Certification Laboratory Testing Procedures Histopathology Histopathology: Techniques and Formulae Transmission and Scanning Electron Microscopy Bacteriology Mycology Parasitology Parasitology: Techniques and Formulae Virology Serology

In this chapter, the biology (morphology, life cycle), classification, phenotypic and antigenic traits, geographical distribution and host range, diagnosis, economic importance, control, treatment and epizootiology of Viral haemorrhagic septicaemia in fishes with particular emphasis on cultured rainbow trout in the European Union were discussed. Recommendations for future studies were also presented.

Methods for the isolation and quantification of infectious pancreatic necrosis virus (IPNV) from ovarian and seminal fluids of Atlantic salmon are described. Both have utility for the non-lethal detection of IPNV in mature broodstock and for research into vertical transmission. Two experiments are described to check the efficiency of an elution method for the removal of IPNV from milt. The isolation rate for ovarian fluid of females was generally higher than that for seminal fluid of males from the same populations. In IPNV milt mixing experiments up to 99.98% of available IPNV adsorbed to Atlantic salmon spermatozoa and 20-100% of virus eluted using a variety of procedures. Titration of virus from naturally infected milt can be useful in estimating the relative vertical transmission risk from male broodstock.

Infectious dose and shedding rates are important parameters to estimate in order to understand the transmission of infectious pancreatic necrosis virus (IPNV). Bath challenge of Atlantic salmon post-smolts was selected as the route of experimental infection as this mimics a major natural route of exposure to IPNV infection. Doses ranging from 10(2) to 10(-4) 50% end-point tissue culture infectious dose (TCID(50)) mL(-1) sea water were used to estimate the minimum infectious dose for a Scottish isolate of IPNV. The minimum dose required to induce infection in Atlantic salmon post-smolts was <10(-1) TCID(50) mL(-1) by bath immersion (4 h at 10 degrees C). The peak shedding rate for IPNV following intraperitoneal challenge using post-smolts was estimated to be 6.8 x 10(3) TCID(50) h(-1) kg(-1) and occurred 11 days post-challenge. This information may be incorporated into mathematical models to increase the understanding of the dispersal of IPNV from marine salmon sites.

A staphylococcal coagglutination (COA) test was compared to an enzyme linked immunosorbant assay (ELISA) for confirming the presence of infectious pancreatic necrosis virus (IPNV) in cell culture supernatant. There was close correlation between the COAtest and ELISA at titres above 2.5x10(5) TCID50/ml. The COA test required little equipment, was quick, simple and reliable in use.

During mid-June 1999 peak mortalities of 11% of the total stock per week were seen at a sea cage site of Atlantic salmon, Salmo salar L., post-smolts in the Shetland Isles, Scotland. Virus was isolated on chinook salmon embryo (CHSE) cells in a standard diagnostic test and infectious pancreatic necrosis virus (IPNV) identified by enzyme-linked immunosorbent assay. IPNV was confirmed as serogroup A by a cell immunofluorescent antibody test using the cross-reactive monoclonal antibody AS-1. Four weeks after the main outbreak, virus titres in surviving moribund fish were assayed at >10(10) TCID50 g(-1) kidney. Histopathology of moribund fish was characterized by pancreatic acinar cell necrosis and a marked catarrhal enteritis of the intestinal mucosa. In the liver, necrosis, leucocytic infiltration and a generalized cell vacuolation were noted. IPNV-specific immunostaining was demonstrated in pancreas, liver, heart, gill and kidney tissue. The nucleotide sequence of the coding region of segment A was determined from the Shetland isolate. A 1180 bp fragment of the VP2 gene of this isolate was compared with a 1979 reference isolate from mainland Scottish Atlantic salmon, La/79 and another more recent mainland isolate, 432/00. Both A2 isolates were derived from carrier fish without signs of IPN and serotyped by a plaque neutralization test. The Shetland isolate shows a different nucleotide and amino acid sequence compared with the two isolates from carrier fish. These latter isolates showed identical amino acid sequences in the fragment examined, despite the 21 years separating the isolations. Sequence comparisons with other A2 (Sp) isolates on the database confirm all three Scottish isolates are A2 (Sp).

Following the infectious salmon anaemia outbreak in Scotland in May 1998, there was a strong interest in evaluating alternatives to sodium hypochlorite for the disinfection of fish fanning equipment. Because no information was available, a programme of laboratory testing of several disinfectants against cultured ISA virus was commenced.Cultured ISA virus was grown on permissive cell lines and a stock stored at -80 degreesC. Disinfectant assays were set up using a procedure similar to that recommended for viruses of higher animals under the UK Animal Health Act (1981). Dilutions of disinfectant at the manufacturer's recommended dose were made in W.H.O. hard water at 4 degreesC and ISA virus was added for 5 min exposure. Neutralizer was added to inactivate the disinfectant and the samples were dialysed. Residual and starting virus was titrated on SHK-1 cells using a focus-forming assay (FFA). Briefly, cell layers were fixed with acetone, incubated with diluted ISA virus monoclonal antibody 3H6F8, then incubated with diluted goat anti-mouse Ig-phosphatase conjugate and bound conjugate visualised with fast red TR/napthol-AS MX stain. Stained foci were counted.

IPNV in carrier Atlantic salmon parr was titrated in the supernatant after homogenisation of the kidney and compared with the titre in the supernatant of the sonicated cell pellet. In 17/20 fish the cell-associated and particulate-bound virus exceeded that in the clarified homogenate supernatant. In two method comparison experiments using pre- or post-smolt field samples, sonicated cell pellet after homogenisation yielded the highest number of isolations.

The TO cell line was tested for the isolation of infectious salmon anaemia virus (ISAV) from Atlantic salmon organs. Isolations were made from stored Liver and mixed organ samples from a confirmed case of ISA in Scotland. TO, SHK-1 and CHSE-214 cells were used. The TO cell line was faster to show a cytopathic effect than SHK-1 or CHSE-214 cells and also isolated virus from more samples than the other cell lines. These results indicate that TO cells can be used to improve the sensitivity of virus isolation tests for Scottish isolates of ISAV.

Journal of Fish DiseasesVolume 26, Issue 5 p. 309-312 Experimental challenge of post-smolts with IPNV: mortalities do not depend on population density T J Bowden, T J Bowden Marine Laboratory, Victoria Road, Aberdeen, Scotland, UKSearch for more papers by this authorK Lockhart, K Lockhart Marine Laboratory, Victoria Road, Aberdeen, Scotland, UKSearch for more papers by this authorD A Smail, D A Smail Marine Laboratory, Victoria Road, Aberdeen, Scotland, UKSearch for more papers by this authorA E Ellis, A E Ellis Marine Laboratory, Victoria Road, Aberdeen, Scotland, UKSearch for more papers by this author T J Bowden, T J Bowden Marine Laboratory, Victoria Road, Aberdeen, Scotland, UKSearch for more papers by this authorK Lockhart, K Lockhart Marine Laboratory, Victoria Road, Aberdeen, Scotland, UKSearch for more papers by this authorD A Smail, D A Smail Marine Laboratory, Victoria Road, Aberdeen, Scotland, UKSearch for more papers by this authorA E Ellis, A E Ellis Marine Laboratory, Victoria Road, Aberdeen, Scotland, UKSearch for more papers by this author First published: 14 May 2003 https://doi.org/10.1046/j.1365-2761.2003.00456.xCitations: 14 Dr A E Ellis, Marine Laboratory, Victoria Road, Aberdeen AB11 9DB, Scotland, UK (e-mail: [email protected]) Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Citing Literature Volume26, Issue5May 2003Pages 309-312 RelatedInformation

Atlantic salmon smolts, previously unexposed to infectious pancreatic necrosis virus (IPNV), were placed into tanks of sea water at 10 degreesC. After 4 weeks, 40 fish were injected intraperitoneally (i.p.) with homogenized and filter-sterilized kidney material obtained from salmon with clinical IPN in a marine farm in Shetland. The injected fish were cohabited with 40 untreated fish. Mortalities began in the injected fish on day 7 and reached a peak of 48% on day 14. In the cohabitation group, mortalities began on day 14 and reached a peak of 70% on day 27. The IPNV in the Shetland kidney homogenate was cultured in Chinook salmon embryo (CHSE) cells and passed twice. This cultured virus was injected i.p. into fish at various doses ranging from 10 to 10(7) TCID50 fish(-1) 4 weeks after seawater transfer. Challenge tanks contained 30 injected fish and 30 cohabitees. Mortality rates and levels were dose-dependent. The highest dose used resulted in a similar mortality pattern as obtained with a similar dose of the Shetland kidney homogenate, indicating that virulence was retained after two passes in tissue culture. Even with the lowest dose, mortality reached 12% in the injected group and 23% in the cohabitees. The IPNV titres were high (10(6) -10(9) i.u. g(-1) kidney) in fish which died during the experiment and low (<10(5) i.u. g(-1) kidney) or undetectable in surviving fish. The cultured virus (pass 3) was used in a challenge model where the population density of fish in the tanks was high (50 injected and 50 cohabitees) or low (15 injected and 15 cohabitees). In the high stocking density tank, mortalities peaked at about 35% in the injected group and at 52% in the cohabitees. In the low stocking density tank, mortalities peaked at about 40% in the injected fish but no mortality occurred in the cohabitees. However, IPNV was detected (up to 10(4) i.u. g(-1) kidney) in 82% of cohabitees sampled on day 30. These data suggest that lethal lateral transmission of the virus is dependent on the infectious pressure from the injected group. A further trial was conducted to investigate the effect of time post-seawater transfer on the susceptibility of post-smolts to IPN. Groups of fish were challenged every 2 weeks from week 0-10. Few mortalities occurred at week 0 and virus titres were high in these fish. Most survivors became carriers, some with titres >10(6) i.u. IPNV g(-1) kidney. From 2 to 10 weeks after seawater transfer, mortalities in both injected and cohabitees were substantial with viral titres >10(7) i.u. g(-1) kidney. Survivors had lower titres and in many virus was undetectable. Throughout the experiments, moribund fish were sampled for histology and all showed typical IPN histopathology.

A Scottish salmon pancreas disease virus (SPDV) has been isolated and its optimum growth conditions determined. Although several fish cell lines have been tested, successful culture was achieved only with CHSE-214 cells. Cytopathic effects were observed after 5 days. The highest virus titres, calculated by microtitration assay, were reached at 15 degrees C. After 7-9 days post-inoculation, CHSE-214 cell supernatants contained between 10(7)-10(5) TCID50 ml(-1) The cultured isolate is chloroform- and pH 3.0-sensitive, and virions are 50-60 nm in diameter. These characteristics are similar to the Irish SPDV isolates. The culture isolate induced typical pancreas disease (PD) lesions in experimentally infected Atlantic salmon and convalescent fish were resistant to experimental infection with PD-infective kidney homogenates obtained by serial in vivo passages from a PD-infected farmed salmon (termed wild-type SPDV). Furthermore, fish immunised with the inactivated cultured virus were protected against a cohabitation challenge with the wild-type virus. Immunised fish sera showed virus-neutralising activity before challenge (7 weeks post-immunisation) and from 3-6 weeks post-challenge, when sera from non-immunised fish did not neutralise the virus. At 6 weeks post-cohabitation challenge, previously immunised fish had neutralising titres of up to 1:65. Following intraperitoneal (i.p.) challenge, immunised fish showed neutralising titres as high as 1:226 at 8 weeks post-challenge. Non-immunised fish injected i.p. with the wild-type virus developed serum-neutralising activity against the cultured isolate when sampled 8 weeks after infection, confirming an antigenic relationship between the wild-type and cultured virus. The results demonstrate that the tissue culture-adapted isolate of SPDV could be successfully used to protect against challenge by the wild-type virus and could therefore have potential use as an inactivated vaccine against PD.

A surveillance programme was initiated on the occurrence and distribution of viral haemorrhagic septicaemia virus (VHSV) in wild marine fish. Six research cruises were undertaken in an 18 mo period during 1997 and 1998, covering the North Sea, the Atlantic waters off the north and west coasts of Scotland and the Irish Sea. A total of 19,293 fish were sampled from 23 different species including cod, haddock, Norway pout, herring and sprat. Individual fish lengths were recorded and the fish were checked for lesions, haemorrhaging and other signs of disease. Pools of organ samples were taken for virus assay. The majority of fish sampled did not display clinical signs indicative of viral haemorrhagic septicaemia. A small number of cod were found with skin lesions and haddock with skin haemorrhaging. Of the 2081 organ and skin sample pools collected, 21 tested positive for VHSV by tissue culture and enzyme-linked immunosorbent assay. Seventeen of the isolates originated from Norway pout Trisopterus esmarkii, one from cod Gadus morhua (skin lesion), one from herring Clupea harengus, one from whiting Merlangius merlangus, and one from a previously unreported host species, poor cod Trisopterus minutus.

A truncated form of the structural protein VP2 (truncVP2) of infectious pancreatic necrosis (IPN) virus encompassing amino acids 147–307 was expressed in bacterial, yeast, piscine and mammalian cells. All four recombinant antigens were recognised by a VP2-specific monoclonal antibody by ELISA and immunoblot analysis. However, the minimum amount of r-truncVP2 needed for detection by these methods varies depending on the cell type used for expression. Furthermore, all four recombinant preparations, when used to immunise Atlantic salmon, were capable of inducing antibodies reactive with whole IPNV in ELISA.

Haemadsorption using a variety of erythrocytes has been used to detect intracellular infection of SHK-1 cells by ISA virus (ISAV), in the absence of a cytopathic effect (CPE). The use of haemadsorption has made possible the detection of ISAV in farmed and wild fish in Scotland in cases where no CPE was detected in SHK-1 cells.

During fish disease surveys for marine rhabdoviruses in 1993 and 1995, the cod ulcus syndrome was seen widely in all ages of cod, especially the 2 to 5+ year classes. Viral Haemorrhagic Septicaemia Virus (VHSV) was isolated from a small proportion of the lesion-positive fish and these isolates were identified by immunofluorescence or ELISA. A serendipitous observation of dermal petechiae on haddock was made. VHSV was isolated from this lesion for the first time indicating a new host species for VHSV.

Juvenile pathogen-free turbot were infected with a viral haemorrhagic septicaemia virus (VHSV) isolate recovered from turbot cultivated on the island of Gigha, West Scotland. Mortality of 100% was recorded in fish infected via the intra-peritoneal (i.p.) route. Horizontal transmission of VHSV in sea water was demonstrated by cohabitation of naive fish with i.p. infected fish at a ratio of 1:1. The total cumulative average mortality in cohabiting fish was 60% by 60 d post-infection. Turbot infected via an immersion route exhibited a cumulative average mortality of 71% by the end of the experiment. VHSV identified by enzyme-linked immunosorbent assay (ELISA) was recovered from both organ (kidney and spleen) and brain samples of individual fish that died following infection by all experimental routes. These findings pose significant implications regarding the persistence of VHSV and its role in limiting natural populations of marine fish species. In addition, the establishment of infection models for the transmission of VHSV in sea water is of fundamental importance to the development of anti-VHSV vaccines in important commercial species such as turbot.