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Opinion of the Panel on Animal Feed of the Norwegian Scientific Committee for Food Safety Risk assessment regarding processing requirements of by-products from aquaculture for use in fish feed Risk assessment regarding processing requirements of by-products from aquaculture for use in fish feed

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Zur völligen Inaktivierung der Viren waren bis zu 540 mg Cl2/l und Einwirkungszeiten zwischen 2 und 20 Minuten notwendig.DanksagungDie Untersuchungen wurden aus Mitteln des Bundesministeriums für Ernährung, Landwirtschaft und Forsten, Bonn, finanziert.Fräulein C. Held sei für die technische Assistenz, Fräulein van de Graaff für die Mitarbeit bei der Durchführung der Chlorbestimmung gedankt.SummaryComparative studies on the stability of four fish-pathogenic viruses (VHSV, PFR, SVCV, IPNV)The effects of environmental conditions (water, mud, drying), the action of gamma and UV irradiation as well as the influence of chemical components and disinfectants on the infectivity of VHSV, PFR, SVCV and IPNV were tested.Storage in tap water (10 °C) reduced virus titres but SVCV survived for 42, VHSV for 49, PFR for 70 and IPNV for more than 231 days.Viruses suspended in mud (pH 7,6, 4 °C) were stable for 10 (VHSV), 42 (PFR, SVCV) and for more than 210 days (IPNV).Drying of the viruses resulted in only inactivation of 1 log TCID50 and survival was not affected by dry condition for more than 28 days at 4 and 20 °C.Gamma irradiation (1000 krad) resulted in inactivation of 90% for IPNV and > 99.99% for VHSV, PFR, SVCV.Ultraviolet irradiation (254 nm) effected a 100% reduction of the infectivity of VHSV, PFR, SVCV in 10 min., of IPNV in 60 min.Methylene blue (20 mg/l), malachite green (10 mg/l) Mefarol® (1%) and copper sulphate (100 mg/l) had no effect on the viral activities of VHSV, PFR, SVCV, IPNV.NaOH (2%) and formalin (3% of the 40% formaldehyde-solution) destroyed all viruses investigated within 5–10 min.Acid (pH 3) reduced the infectivity of VHSV significantly in 3 hours but did not affect the infectivity of IPNV.In order to inactivate the viruses investigated to exposures to chlorine (Cl2) up to 540 mg/l for 2–20 min. were needed.RésuméRecherches comparées sur la stabilité de quatre virus pathogènes du poisson (VHSV, PFR, SVCV, IPNV)Les virus pathogènes du poisson (VHSV, PFR, SVCV, IPNV) se sont montrés relativement stables vis-à-vis de facteurs chimico-physiques. SVCV est resté infectieux dans l'eau à 10 °C durant 42 jours, VHSV 49 jours, PFR 70 jours et IPNV > 231 jours.VHSV a survécu à 4 °C dans de la boue d'étang (pH 6,79) 10 jours, PFR et SVCV 42 jours, IPNV plus de 210 jours.Les virus examinés sont restés infectieux plus longtemps que 28 jours à l'état desséché aussi bien à 4 qu'à 20 °C.Les raysons γ (1000 krad) ont inactivé complètement les virus encapsulés (VHSV, PFR, SVCV) et partiellement (IPNV (∼ 90%).Les Rhabdovirus (VHSV, PFR, SVCV) ont été complètement inactivés en 10 minutes avec des rayons UV (254 nm) et le virus non capsulé IPN en 60 minutes.Le bleu de méthylène (20 mg/l), le vert de malachite (10 mg/l), Mefarol® (1%) et le sulfate de cuivre (100 mg/l) n'ont pas eu d'effet d'inactivation sur les virus examinés.NaOH (2%) et la formaline (3% d'une solution à 40%) ont par contre complètement inactivé les agents en 5–10 minutes.L'acidité (pH 3) a inactivé le Rhabdovirus VHSV encapsulé en 3 heures, mais n'a pas eu d'influence sur l'infectiosité d'IPNV encapsulé après 6 heures.Une solution d'hypochlorite de sodium a inactivé les virus examinés en utilisant partiellement en remplacement des concentrations en Cl2.Une concentration jusqu'à 540 mg Cl2/l et des temps d'action entre 2 et 20 minutes ont été nécessaires pour une inactivation complète de virus.ResumenEstudios comparados sobre la estabilidad de cuatro virus piscipatógenos (VHSV, PFR, SVCV e IPNV)Los virus piscipatógenos (VHSV, PFR, SVCV e IPNV) se mostraron como relativamente estables frente a los influjos físicoquímicos.En el agua (10 °C) permaneció el SVCV 42 días infeccioso, mientras que el VHSV 49, el PFR 70 y el IPNV más de 231 días.En el barro de la laguna (pH 6.79) sobrevivieron a 4 °C el VHSV 10 días, el PFR y el SVCV 42 y el IPNV más de 210 días.Al estado desecado conservaron los virus examinados la infecciosidad tanto a 4° como a 20 °C durante más de 28 días.Los rayos γ (1.000 Krad) inactivaron los virus con envoltura (VHSV, PFR y SVCV) por completo, y el IPNV solo parcialmente (∼ 90%).Por medio de los rayos UV (254 nm) se inactivaron por completo los rabdovirus (VHSV, PFR y SVCV) en un plazo de 10 minutos, mientras que el virus IPN, sin envoltura, antes de 60 minutos.El azul de metileno (20 mg/l), verde de malaquita (10 mg/l), Mefarol® (1%) y sulfato de cobre (100 mg/l) no ejercieron ninguna acción inactivadora sobre los virus investigados.La lejía de hidróxido sódico (2%) y el aldehido metílico (3% de solución de aldehido fórmico al 40%) inactivaron por completo los agentes etiológicos en un plazo de 5–10 minutos.El medio ácido (pH 3) inactiva, eso sí, el rabdovirus VHSV, provisto de envoltura, en un plazo de tres horas, pero no ejercía ningún influjo sobre la infecciosidad del IPNV, que no tiene envoltura, incluso después de 6 horas.La solución de hipoclorito sódico inactivaba solo parcialmente los virus examinados, empleando concentraciones admisibles de Cl2. 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Chapter
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The purpose of this study was to evaluate methods for inactivation of fish pathogens in outlet water from fish slaughteries. The effect of three disinfectants, namely sodiumhypochlorite, heat and photozone, were tested on two fish pathogens, Vibrio anguillarum and Yersinia ruckeri. Outlet water was incubated with the test bacteria for 24 h prior to use, and contained in the area of 106–109 CFU (Colony Forming Units) per ml.The following doses gave 100% inactivation of the test bacteria: 30 min exposure to 250 ppm sodiumhypochlorite, 60°C for 2 min, 72°C for 15 s and a photozone generated redox potential of approximately 550 mV for 30 min.The following doses resulted in 100% inactivation of all bacteria in the outlet water: 30 min exposure to 350 ppm sodiumhypochlorite, 72°C for 15 s and a photozone generated redox potential of approximately 550 mV for 30 min.
Article
Aquatic birnaviruses infect a large variety of fish, molluscs and crustacea in the freshwater, estuarine and marine environments throughout the world and yet despite this extensive host range and geographical distribution, the vast majority of isolates have been found to be antigenically-related to the original reference serotypes (VR299, Sp and Ab) of infectious pancreatic necrosis virus (IPNV) of salmonids. However, in early studies it was seen that there is a high degree of antigenic diversity amongst isolates with some reacting only relatively weakly with these three traditional serotypes, suggesting that other serotypes may exist. In this review we examine the published studies on the degrees of antigenic relatedness between IPNV isolates and other aquatic birnaviruses and discuss the attempts by various authors to identify and/or define different serotypes. The published data from serum neutralization tests is critically appraised and the factors influencing neutralization results discussed. Details are then presented of our own cross-neutralization study with almost 200 isolates of IPNV and other aquatic birnaviruses using a standardized procedure. On the basis of the limit we set for the maximum degree of difference from a reference serotype for an isolate to belong to that serotype, the isolates we examined could be divided into two distinct serogroups (which do not cross-react), the first containing nine serotypes and the second containing a single serotype. We propose a change from the traditional terminology of the recognised reference serotypes to a new simplified system: Serogroup A containing serotypes A1 to A9 (so far), and Serogroup B containing the single serotype B1 (so far). The use of panels of selected monoclonal antibodies against one or more of these reference serotypes offers the prospect for a simpler method for serotyping new isolates and identifying new serotypes. In limited studies to date, reaction patterns with panels of monoclonal antibodies in immunodot and ELISA tests have, in the main, substantiated the serotype classification scheme achieved by serum neutralization tests.
Article
The thermal and pH resistance of five representative salmonid fish pathogens were tested to examine the efficacy of procedures currently used for processing fish viscera to be incorporated into fish diets. Aeromonas salmonicida, Mycobacterium chelonei, Renibacterium salmoninarum, infectious hematopoietic necrosis virus (IHNV) and infectious pancreatic necrosis virus (IPNV) were suspended in buffered medium at pH 7.0 or 4.0, or in fish silage at pH 4.0 and their survival at selected temperatures was determined. When suspended in medium at pH 7.0, A. salmonicida, M. chelonei and IHNV were sensitive to temperatures in a range of 40–60°C. Renibacterium salmoninarum and IPNV were both more resistant to that temperature range in the neutral pH medium; however, each was inactivated by conditions simulating pasteurization. The combination of heat and low pH currently used for the processing and pasteurization of fish viscera should ensure that the resulting fish food is safe against spreading infectious diseases.
Article
  The Vibrionaceae are environmentally ubiquitous to estuarine waters. Two species in particular, V. vulnificus and V. parahaemolyticus, are important human pathogens that are transmitted by the consumption of contaminated molluscan shellfish. This document provides a comprehensive review of the current state of knowledge about these important foodborne disease agents. Topics include the epidemiology of human disease; biotypes and virulence factors; cultural and molecular-based detection methods; phenotyping and genotyping approaches; microbial ecology; and candidate control strategies. Recent international risk assessment efforts are also described. The reader will gain an understanding of why these organisms pose a public health risk and how improving our understanding of their behavior in the environment and the host can aid in reducing that risk in the future.
Article
The effects of some chemical disinfectants, organic solvents, hydrogen ions, heat, ultraviolet (UV) irradiation and ozone on the inactivation of striped jack nervous necrosis virus (SJNNV) were investigated. SJNNV was inactivated by contact with a final concentration of 50 ppm of sodium hypochlorite, calcium hypochlorite, benzalkonium chloride and iodine for 10 min at 20 °C. Cresol concentrations of more than 10 000 ppm were required to inactivate SJNNV, and no inactivation of SJNNV by formalin was detected at any concentration tested. The effective concentrations of ethanol and methanol were 60% and 50%, respectively, but SJNNV was resistant to ether and chloroform. SJNNV was inactivated by high alkalinity, pH 12 for 10 min at 20 °C, and also inactivated by heat treatment at 60 °C for 30 min. Inactivation of SJNNV by UV irradiation was observed at an intensity of 410 μW cm−2 for 244 s. Ozone at 0.1 μg ml−1 as a total residual oxidant was required to inactivate SJNNV for 2.5 min. Washing fertilized eggs and the treatment of sea water with ozone decreased the rate of occurrence of VNN.
Article
Bacterial and viral fish pathogens were exposed to ozone or ultraviolet (UV) irradiation in laboratory batch systems. Inactivation curves were made for Aeromonas salmonicida subsp. salmonicida, Vibrio anguillarum, Vibrio salmonicida, Yersinia ruckeri and the infectious pancreatic necrosis virus (IPNV) in ozonated lake, brackish and sea water at 9–12°C. The four bacteria tested were inactivated by 99·99% (4 log reductions in viable count) in all three waters within 180s at residual ozone concentrations of 0·15-0·20 mg/liter measured by the indigo colorimetric method. After establishing these residuals, the differences in water salinity did not cause any substantial differences in bactericidal activity of ozone, illustrating the usefulness of concentration measurements by the indigo colorimetric method to predict inactivation also in saline waters. The rate of bacterial inactivation was fast during the first 60 s in all three waters. After that point the slope of the curves levelled off. This observation was explained by loss of ozone and reduced bactericidal activity during the course of the experiments. IPNV was inactivated (99·99%) in all three waters within 60 s when exposed to 0·10-0·20 mg/liter residual ozone.V. anguillarum, V. salmonicida, Y. ruckeri and IPNV were UV irradiated in brackish water at room temperature. An UV dose of 2·7 m Ws/cm2 resulted in 99·999% (5 log) reduction in viable count for all three bacteria. IPNV was much more resistant to irradiation than the bacteria. An average UV dose of 122 mWs/cm2 was required for 99·9% (3 log) reduction in virus titer.
Article
Infectious pancreatic necrosis virus (IPNV) (serotype Sp) was exposed to temperatures between 60 and 90°C in a medium mimicking the water-soluble phase of hydrolyzed fish by-products. D values ranged from 290 to 0.5 min, and the z value was approximately 9.8°C. Addition of formic acid to create a pH 4 medium did not enhance heat inactivation. Predicted inactivation effects at different temperature-time combinations are provided.
Article
Moritella viscosa is the causative agent of winter ulcer disease of marine fish. Knowledge of its pathogenicity is limited and there are no reports comparing the virulence properties of a collection of bacterial isolates. The in vivo and in vitro virulence of the extracellular products (ECP) of 22 M. viscosa isolates was screened. Two non-virulent Canadian isolates and a Norwegian isolate with reduced virulence produced non-lethal ECP. Correlation was obtained between cytotoxin and haemolysin production of M. viscosa. Isolates from salmon produced ECP with lower cytotoxic and haemolytic activities than ECP of isolates originating from other hosts. Correlation was not found between lethality of ECPs in salmon and cytotoxic or haemolytic activities. All isolates secreted esterases and a metallopeptidase (MvP1), degraded starch and produced siderophores. Variable levels of ECP protein concentration, different enzymatic activities and siderophore production could not explain differences in virulence. The results show that virulent M. viscosa isolates secrete a lethal toxic factor of unknown nature and that cytotoxin production may reflect host adaptation. Cell-culture models may not be optimal for determining the virulence of M. viscosa, as no association between cytotoxicity and bacterial virulence was obtained. Non-virulent strains may be useful in future research on M. viscosa virulence, as construction of mutants has not been successful.
Article
A series of recent reports have implicated bacteria from the family Francisellaceae as the cause of disease in farmed and wild fish and shellfish species such as Atlantic cod, Gadus morhua L., tilapia, Oreochromis spp., Atlantic salmon, Salmo salar L., three-line grunt, Parapristipoma trilineatum (Thunberg), ornamental cichlid species, hybrid striped bass Morone chrysops x M. saxatilis and, recently, a shellfish species, the giant abalone, Haliotisgigantea Gmelin. The range of taxa affected will very probably rise as it is likely that there has been considerable under-reporting to date of these disease agents. In common with other Francisella species, their isolation and culture require specialized solid and liquid media containing cysteine and a source of iron. This likely restricted earlier efforts to identify them correctly as the cause of disease in aquatic animals. The most information to date relates to disease in cod, caused by F. noatunensis and tilapia, caused by F. noatunensis subsp. orientalis (also termed F. asiatica), both causing granulomatous inflammatory reactions. Mortalities in both species can be high and, as the disease can likely be transferred via live fish movements, they pose a significant threat to tilapia and cod aquaculture operations. Although the fish-pathogenic Francisella species are classified in the same genus as the human pathogens F. tularensis, causative agent of tularemia, and F. philomiragia, the risk to humans from the fish and shellfish pathogenic Francisella species is considered very low.
Article
A cohort study was initiated in the spring of 2006 to investigate epidemiological aspects and pathogenesis of salmonid alphavirus (SAV) subtype 3 infections and pancreas disease (PD). The aims were to assess involvement of the freshwater production phase, the extent and frequency of subclinical infections and to follow PD-affected populations throughout the entire seawater production cycle, as well as investigate possible risk factors for PD outbreaks. Fish groups from 46 different Atlantic salmon freshwater sites in six counties were sampled once prior to seawater transfer and followed onto their seawater sites. A total of 51 Atlantic salmon seawater sites were included, and fish groups were sampled three times during the seawater production phase. SAV subtype 3 was not identified by real-time RT-PCR from samples collected in the freshwater phase, nor were any SAV-neutralizing antibodies or histopathological changes consistent with PD. In the seawater phase, SAV was detected in samples from 23 of 36 (63.9%) studied sites located within the endemic region. No SAV subtype 3 was detected in samples from seawater sites located outside the endemic region. The cumulative incidence of PD during the production cycle amongst sites with SAV detected was 87% (20 of 23 sites). Average fish weight at time of PD diagnosis ranged from 461 to 5978 g, because of a wide variation in the timing of disease occurrence throughout the production cycle. Mortality levels following a PD diagnosis varied greatly between populations. The mean percentage mortality was 6.9% (+/-7.06) (range 0.7-26.9), while the mean duration of increased mortality following PD diagnosis was 2.8 months (+/-1.11) (range 1-6).
Article
Disease associated with salmonid Alphavirus (SAV) infection is a significant problem for farm production of salmonids in Europe. The SAV subtype 3 (SAV3) is a Norwegian subtype present exclusively in production systems for Atlantic salmon Salmo salar and rainbow trout Oncorhynchus mykiss in western Norway. It has been suggested that SAV3 is transmitted through smolt transport from the main area for SAV disease in western Norway to as far as northern Norway. One explanation for this type of spread is that SAV is present at freshwater production sites for Atlantic salmon smolts. The present study confirms this, showing that SAV3 is present at smolt production sites in Norway. At two sites in northern Norway that had received eggs from broodfish companies in Hordaland County, western Norway, 2-4-g fry were positive for SAV3. Hence, it cannot be excluded that vertical transmission could have contributed to the presence of SAV3 in northern Norway. In the present study, we followed the normal production cycle for Atlantic salmon in a fish farming company in Hordaland County. Twelve of 353 broodfish in study 1 and 28 of 31 broodfish in study 2 were found to be carriers of SAV3. In the same two studies, SAV was also detected in eggs (1 of 220), eyed eggs (3 of 270), and fry (6 of 600). The SAV was not detected in parr, smolts, or postsmolts, but after a year at sea the fish developed SAV disease. Given the difficulties in tracing the virus through the production cycle until development of SAV disease in the marine farm, we cannot draw any firm conclusions about whether vertical transmission occurs in Norwegian salmon production, and we cannot exclude the possibility that the development of SAV after 1 year at sea was caused by horizontal transmission rather than vertical transmission.
Article
The infection route of the marine fish pathogen Vibrio anguillarum was studied after oral challenge of the juvenile turbot Scophthalmus maximus L. through a life feed. Artemia nauplii were incubated in a suspension of V. anguillarum cells, and subsequently fed twice to the fish. All challenged fish died within 4d after the first challenge, while no mortality occurred in the non-challenged controls. The results of an immunohistochemical examination of the sectioned fish samples clearly demonstrated that V. anguillarum cells were ingested by the Artemia and that the latter were ingested by the fish. Bacteria were released from the Artemia mainly in the anterior part of the intestine. Most challenged fish started to show disease signs 24h after the second challenge and died 2d later. A histopathological analysis of moribund fish showed the development of septicaemia. Moreover, the sequential sampling, allowed the reconstruction of the infection route after oral challenge. Our results show that V. anguillarum was transported through the intestinal epithelium by endocytosis, after which the bacterium was released in the lamina propria. From there the bacterium was transported by the blood to the different organs, eventually leading the septicaemia and mortality.
Article
Vibrios are Gram-negative rod-shaped bacteria that are widespread in the coastal and estuarine environments. Some species, e.g. Vibrio anguillarum and V. tapetis, comprise serious pathogens of aquatic vertebrates or invertebrates. Other groups, including Grimontia (=Vibrio) hollisae, Photobacterium (=Vibrio) damselae subsp. damselae, V. alginolyticus, V. harveyi (=V. carchariae), V. cholerae, V. fluvialis, V. furnissii, V. metschnikovii, V. mimicus, V. parahaemolyticus and V. vulnificus, may cause disease in both aquatic animals and humans. The human outbreaks, although low in number, typically involve wound infections and gastro-intestinal disease often with watery diarrhoea. In a minority of cases, for example V. vulnificus, there is good evidence to actually associate human infections with diseased animals. In other cases, the link is certainly feasible but hard evidence is mostly lacking.
Article
Francisella tularensis is a gram-negative bacterium that can cause gastrointestinal or oropharyngeal tularemia from ingestion of contaminated food or water. Despite the potential for accidental or intentional contamination of foods with F. tularensis, little information exists on the thermal stability of this organism in food matrices. In the present study, the thermal resistance of the live vaccine strain of F. tularensis in four food products (liquid infant formula, apple juice, mango juice, and orange juice) was investigated. D-values ranged from 12 s (57.5 degrees C) to 580 s (50 degrees C) in infant formula with a z-value of 4.37 degrees C. D-values in apple juice ranged from 8 s (57.5 degrees C) to 59 s (50 degrees C) with a z-value of 9.17 degrees C. The live vaccine strain did not survive at temperatures above 55 degrees C in mango juice and orange juice (>6-log inactivation). D-values at 55 to 47.5 degrees C were 15 to 59 s in mango juice and 16 to 105 s in orange juice with z-values of 9.28 and 12.30 degrees C, respectively. These results indicate that current pasteurization parameters used for destroying common foodborne bacterial pathogens are adequate for eliminating F. tularensis in the four foods tested. This study is the first to determine thermal inactivation of F. tularensis in specific foods and will permit comparisons with the thermal inactivation data of other more traditional foodborne pathogens.
Article
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.
Article
Sequence data were generated for portions of the E2 and nsP3 genes of 48 salmonid alphaviruses from farmed Atlantic salmon (AS), Salmo salar L., and rainbow trout (RT), Oncorhynchus mykiss (Walbaum), in marine and freshwater environments, respectively, from the Republic of Ireland, Northern Ireland, England, Scotland, Norway, France, Italy and Spain between 1991 and 2007. Based on these sequences, and those of six previously published reference strains, phylogenetic trees were constructed using the parsimony method. Trees generated with both gene segments were similar. Clades corresponding to the three previously recognized subtypes were generated and in addition, two further new clades of viruses were identified. A single further strain (F96-1045) was found to be distinct from all of the other strains in the study. The percentage of nucleotide divergence within clades was generally low (0-4.8% for E2, 0-6.6% for nsP3). Interclade divergence tended to be higher (3.4-19.7% for E2, 6.5-28.1% for nsP3). Based on these results and using current SAV terminology, the two new clades and F96-1045 were termed SAV subtypes 4, 5 and 6, respectively. SAV4 contained AS strains from Ireland and Scotland, while SAV5 contained only Scottish AS strains. Recently identified SAV strains from RT in Italy and Spain were shown to belong to SAV2. In addition, marine AS strains belonging to SAV2 were identified for the first time. Analysis of the origin of several clusters of strains with identical E2 and nsP3 sequences strongly support horizontal transmission of virus between farms and aquaculture companies. Evidence in support of vertical transmission was not found.
Article
Humphrey, T.J. 1990. Heat resistance in Salmonella enteritidis phage type 4: the influence of storage temperatures before heating. Journal of Applied Bacteriology69 493–497. Storage of cultures of Salmonella enteritidis PT4 at either 4° or 8°C before heating significantly increased heat sensitivity. The differences between fresh and stored cultures, which became apparent after 4–7 h, were more pronounced with cultures stored at the lower temperature and in those heated at 60° rather than 55°C. Incubation of the stored cultures in either egg or Lemco broth for 30 min at 37°C prior to heating enabled the organisms to recover heat resistance.