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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.

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.

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.

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.

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.

Summary The enzyme-linked immunosorbent assay (ELISA) was used to demonstrate the presence of infectious pancreatic necrosis virus (IPNV) antigen in cell cultures and in fish. Virus antigen could be detected in infected cell cultures before visible cytopathic effect (c.p.e.) was evident and cell cultures showing complete viral c.p.e. produced intense colour reactions. Virus antigen was detected in infected fry during and immediately after an epizootic, but although IPNV-carrier fish could be detected by the ELISA technique, the sensitivity of detection was not as great as that of isolation of the infectious virus in cell culture. The major IPNV serotypes, Sp, Ab and Vr, cross-reacted at only a low level and it was shown that the ELISA technique could be used to serotype IPNV strains rapidly. None of 10 other fish pathogenic viruses reacted with plates sensitized for IPNV detection. The time taken to perform the technique was reduced to 1 h 35 min at room temperature and this still allowed the results to be readily assessed visually as antigen-positive or antigen-negative.

Although the complex series of events which occur when a steroid hormone is administered to a human subject or to an intact animal might reflect a series of completely unre!lated activities, they are more likely to be secondary to a few fundamental actions on the celils of the body and it is with this second concept that research in this field has been directed. There are two particular features of steroids which may be important in determining their mode of action. Firstly, they have oxygen substituents at certain typical positions which readily undergo enzymatic oxido-reduction and could, therefore, act as coenzymes or prosthetic groups of enzymes in reactions involving hydrogen transfer. Secondly, steroids are surface active and interact with hydrophobic surfaces producing energy. If the energy were to be absorbed iby a receptor molecule it could modify the structure and hence the biological activity of that molecule. A less well defined change is associated with the ability of steroids to capture electrons; this has recently been measured by Lovelock, Simmonds and Vandenheuvel (1963) who consider that the high electron affinities of adrenocortical hormones, a property unusual among organic compounds, might indicate their ability to participate in or control biological oxidative processes. Laidler and Krupka (1961) compared the association between the steroid and a receptor with the formation of an activated enzymesubstrate Michaelis complex. Entropy and volume changes during activation of enzymes indicate that structural changes occur in the enzyme molecule and such changes might explain the disturbances of membrane permeability in nerve cells associated with structural changes in acetylcholinesterase. A similar mechanism might account for changes induced by steroids in the permeability of cell structures. In this process parts of the receptor molecules having specific binding properties might be masked, unmasked, created or destroyed; as well as causing a redistribution of bound substrates, an alteration in enzymic properties might result. Kimberg and Yielding (1962) studied the inhibition of pyruvate kinase by oestrogens and related compounds and showed by viscometry and electrophoresis structural changes in the enzyme, without a change in molecular weight. Yielding and Tomkins (1962) have reported a steroid-hormone induced loss of activity of crystalline glutamic dehydrogenase due to disaggregation into subunits functioning as alanine dehydrogenase with an uncovering of pyridine-nucleotide binding sites. These authors have been more concerned to show the possibility of such changes rather than to attach great physiological significance to them. Chemical changes in receptor molecules after association could account for the highly theoretical possibility of the formation of active enzymes from inactive precursors. That the receptor itsel-f could be an enzyme cofactor has been considered by Scott and Engel (1961) who obtained physical evidence for the interaction between steroid hormones and purine dinucleotides. This interaction between hormone and a coenzyme associated with a postulated change in the structure of the coenzyme might abolish its hydrogencarrying function, but there was no experimental evidence for this. Apart from oxido-reductive changes, metabolism of the steroid molecule is not thought to be of physiological importance except as a mechanism for steroid inactivation. However, competition for active sites on enzymes as opposed to association with them could be of importance in influencing steroid metabolism itself. The various theories which have been proposed to explain the action of steroid hormones at the cellular level will be considered under the following headings: 1. Effects on membrane permeability and active transport. 2. Effects on hydrogen transfer. 3. Effects on enzyme induction and protein synthesis.

SUMMARY In order to determine the probable availability of protein-bound cortisol to the tissues, the establishment of equilibrium between transcortin-bound [3H]cortisol and unbound [14C]cortisol in plasma has been followed using Sephadex gel filtration. Association of cortisol with transcortin was rapid; dissociation of the steroid-protein complex was comparatively slow; when the binding-sites were nearly saturated the half-dissociation times of [3H]cortisol were 25 min. at 4°, 1 min. at 22° and about 10 sec. at 37°.